RU2800778C2 - Use of high-therm-resistant cas-protein and a method and a set of reagents for detection of a target nucleic acid molecule - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a system comprising the following: (a) Cas12b protein; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; and (c) a nucleic acid probe that is single stranded DNA. Also the following is disclosed: a device that includes: (1) amplification reaction module — bypass cleavage reactions, and (2) signal detection module.

EFFECT: detecting the presence of target nucleic acid molecules in a sample, as well as detecting SNPs (single nucleotide polymorphisms).

24 cl, 27 dwg, 3 tbl, 11 ex

Description

Technical field

The present invention relates to the field of biotechnology. More specifically, the present invention relates to the use of a highly thermally stable Cas protein and a method and kit for detecting a target nucleic acid molecule.

State of the art

The rapid detection of nucleic acid molecules is a technology that plays a critical role in healthcare, environmental monitoring, criminal investigation and other fields. The detection of nucleic acid molecules can be used not only to detect infestation of humans, animals, plants, etc. pathogens, but also to identify the risks of genetic or oncological diseases, to provide supporting reference materials for individual medicinal products, and to detect microbial contamination of water bodies. Currently, many nucleic acid detection methods have been developed, such as real-time PCR, FISH (fluorescent in situ hybridization) hybridization method, etc. However, there is still a need to develop fast, cheap and sensitive nucleic acid detection methods.

In recent years, the genome editing method based on the CRISPR system (short palindromic cluster repeats) has proven itself well. This task is typically performed by DNA (or RNA) targeted endonucleases such as Cas9. Those skilled in the art can direct Cas9 and other proteins to any target nucleic acid sequence containing a protospacer adjacent motif (PAM) (e.g., Cas9's PAM sequence is NGG) simply by constructing a guide RNA and causing a DNA double-strand break (or RNA cleavage).

In addition to editing genomes, CRISPR can also be used for regulation of gene action, epigenetic editing, functional gene screening, and genomic imaging. Recently, CRISPR, a feature-rich toolkit, has begun to emerge in the field of nucleic acid detection and has attracted attention.

The first method for nucleic acid detection based on the CRISPR system was based on the dCas9 ("dead" Cas9) protein. dCas9 is a Cas9 mutation with two mutated catalytic cleavage active sites. Thus, double-stranded cleavage activity is lost, but dCas9 retains the ability to induce guide RNA and bind to a specific DNA site. Chen et al used dCas9 to hybridize with enhanced green fluorescent protein (EGFP), which can be induced by a specific guide RNA and fluorescence on the target sequence can be observed under a microscope. In 2017, Guk et al reported a dCas9/sgRNA-SG I DNA-FISH system that can selectively detect methicillin-resistant Staphylococcus aureus .

In 2013, Collins and his team created a way to detect the Zika virus on a piece of paper. This method utilizes RNA extraction, amplification and detection reactions based on the Toehold approach. By design, if one strain of the virus differs from another strain in the sequence of the PAM site (i.e. one is NGG and the other is different from it), strains containing PAM can become a target and can be cleaved by the Cas9 protein, thus affecting amplification and detection process based on the Toehold approach.

Recently, the core of another Cas9-based nucleic acid detection method is CRISPR/Cas9-induced isothermal amplification, which can also be used for site-specific nucleic acid detection. This method uses CRISPR/Cas9 cleavage, nickase and DNA polymerase. This method allows not only to detect the target concentration of 0.82 mol, but also to recognize the difference in one base in the nucleic acid molecules. Target recognition in the present method is more versatile than in the previous method.

The essence of another method for detecting nucleic acids is based on the effect of "trans-cleavage" (or "bypass cleavage") of some Cas proteins. In 2016, Zhang Feng et al found that the Cas13a protein (formerly known as C2c2) has a bypass degrading activity. In other words, when Cas13a binds to a target RNA sequence, it exhibits the characteristics of randomly cutting other RNAs, which is used to detect a specific nucleic acid, and this method is called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).

While there are many CRISPR-based nucleic acid detection methods, there is still a need to develop faster, simpler, and cheaper nucleic acid detection methods in this area.

The essence of the invention

The aim of the present invention is to develop faster, simpler and cheaper nucleic acid detection methods based on the CRISPR system.

The first aspect of the present invention is to provide a system for detecting target nucleic acid molecules, which includes:

(a) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; And

(c) a nucleic acid probe that is single stranded DNA;

wherein the target nucleic acid molecule is a target DNA.

In another preferred embodiment, the detection system includes qualitative detection or quantitative detection.

In another preferred embodiment, the detection system further comprises (d) a buffer.

In another preferred embodiment, the detection system further comprises target nucleic acid molecules to be detected.

In another preferred embodiment, the detection system further comprises:

(e1) a polymerase that is used to amplify the target DNA;

(e2) an optional reverse transcriptase which is used for reverse transcription;

(e3) dNTP, which is used for the amplification reaction and/or the reverse transcription reaction.

In another preferred embodiment, the detection system further comprises reagents used for the Loop Isothermal Amplification (LAMP) reaction.

In another preferred embodiment, the concentration of target nucleic acid molecules to be detected in the detection system is from 1×10 ^-9 nM to 1×10 ³ nM; preferably from 1×10 ^-8 nm to 1×10 ² nm.

In another preferred embodiment, the concentration of target nucleic acid molecules to be detected in the detection system is from 1 to 100 copies/µl or from 1 to 1×10 ¹⁵ copies/µl, preferably from 1 to 10 copies/µl, more preferably from 1 up to 5 copies/µl.

In another preferred embodiment, in the detection system, the molar ratio of the nucleic acid probe to the target nucleic acid molecule is from 10 ³ :1 to 10 ¹⁴ :1, preferably from 10 ⁴ :1 to 10 ⁷ :1.

In another preferred embodiment, the length of the guide RNA is 16 to 25 nt, preferably 16 to 22 nt, more preferably 16 to 20 nt.

In another preferred embodiment, the length of the guide RNA is 18 to 25 nt, preferably 18 to 22 nt, more preferably 18 to 20 nt.

In another preferred embodiment, when the target nucleic acid molecule is single-stranded DNA, the site of detection of the target nucleic acid molecule is at position 9 or positions 10-16, preferably at positions 10-14, more preferably at positions 10-12 behind the PAM guide sequence. RNA.

In another preferred embodiment, when the detection site is at position 9 downstream of the guide RNA PAM sequence, the detection site is G.

In another preferred embodiment, the detection site is at positions 9-20, preferably at positions 10-18, more preferably at positions 10-16 behind the PAM guide RNA sequence.

In another preferred embodiment, the site of detection of the target nucleic acid molecule is at positions 1-12, more preferably at position 1, 3 or 10, downstream of the PAM guide RNA sequence.

In another preferred embodiment, the length of the guide RNA is 15-30 nt, preferably 15-18 nt.

In another preferred embodiment, the target DNA comprises DNA derived from reverse transcription of RNA.

In another preferred embodiment, the target DNA contains cDNA.

In another preferred embodiment, the target DNA is selected from the group consisting of single stranded DNA, double stranded DNA, and combinations thereof.

In another preferred embodiment, the nucleic acid probe has a fluorescent group and a quench group.

In another preferred embodiment, both the fluorescent group and the quench group are independently located at the 5' end, 3' end and midpoint of the nucleic acid probe.

In another preferred embodiment, the length of the nucleic acid probe is 3 to 300 nt, preferably 5 to 100 nt, more preferably 6 to 50 nt, most preferably 8 to 20 nt.

In another preferred embodiment, the target nucleic acid molecules comprise target nucleic acid molecules derived from a species selected from the group consisting of: plant, animal, insect, microorganism, virus, and combinations thereof.

In another preferred embodiment, the target DNA is synthetic or naturally occurring DNA.

In another preferred embodiment, the target DNA comprises wild-type or mutant DNA.

In another preferred embodiment, the target DNA comprises DNA obtained by RNA reverse transcription or amplification, such as cDNA and the like.

In another preferred embodiment, the Cas12b protein is selected from the group consisting of: AacCas12b ( Alicyclobacillus acidoterrestris ), Aac2Cas12b ( Alicyclobacillus acidiphilus ), AkaCas12b ( Alicyclobacillus kakegawensis ), AmaCas12b ( Alicyclobacillus macrosporangiidus ), AheCas12b ( Alicyclobacillus herbarius ), and AcoCas12b (Alicyclobacillus contaminans ).

In another preferred embodiment, the nucleic acid probe comprises a single stranded DNA with a detectable label.

In another preferred embodiment, the single stranded DNA is single stranded DNA labeled with a fluorescent label and biotin.

In another preferred embodiment, the single stranded DNA is single stranded DNA labeled with a fluorescent label.

In another preferred embodiment, the single stranded DNA is a fluorescent probe labeled with a fluorescent HEX group at the 5' end and a BHQ1 quench group at the 3' end.

The second aspect of the present invention is to provide a detection system for SNPs (single nucleotide polymorphisms) or nucleotide mutations (containing single-base or multi-base mutations), the system comprising a first detection system and a second detection system;

wherein the first detection system includes:

(a1) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(b1) a first guide RNA, wherein the first guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; And

(c1) a nucleic acid probe that is single stranded DNA;

and the second detection system includes:

(a2) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(b2) a second guide RNA, wherein the second guide RNA guides the Cas12b protein for specific binding to target nucleic acid molecules; And

(c2) a nucleic acid probe that is single stranded DNA;

wherein the target nucleic acid molecule is a target DNA;

in addition, the first guide RNA and the second guide RNA target the same region of the nucleic acid sequence containing the SNP site, and the first guide RNA targets the wild-type (or unmutated) nucleic acid sequence of the SNP site, and the second guide RNA targets to the mutated nucleic acid sequence of the SNP site.

In another preferred embodiment, the detection system also includes a blank sample system.

In another preferred embodiment, the blank sample system includes:

(a3) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(c3) a nucleic acid probe that is single stranded DNA.

In another preferred embodiment, the SNP site is at position 9 or at positions 10-16, preferably at positions 10-14, more preferably at positions 10-12 behind the PAM guide RNA sequence.

In another preferred embodiment, when the SNP site is at position 9 downstream of the guide RNA PAM sequence and the site of detection is G.

In another preferred embodiment, the SNP site is located at positions 9-20, preferably at positions 10-18, more preferably at positions 10-16 behind the PAM guide RNA sequence.

In another preferred embodiment, the length of the first or second guide RNA is 16 to 25 nt, preferably 16 to 22 nt, more preferably 16 to 20 nt.

In another preferred embodiment, the length of the first or second guide RNA is 18 to 25 nt, preferably 18 to 22 nt, more preferably 18 to 20 nt.

The third aspect of the present invention is to provide a kit for the detection of target nucleic acid molecules, including:

i) a first container and a Cas12b protein contained in the first container, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

ii) an optional second container and a second guide RNA contained in the second container, the second guide RNA directing the Cas12b protein for specific binding to target nucleic acid molecules;

iii) a third container and a nucleic acid probe contained in the third container;

iv) an optional fourth container and a buffer located in the fourth container;

wherein the target nucleic acid molecule is a target DNA.

In another preferred embodiment, any two, three or four (or all) of the first, second, third and fourth containers may be the same container or different containers.

In another preferred embodiment, the nucleic acid probe has a fluorescent group and a quench group.

In another preferred embodiment, the kit further comprises a buffer.

In another preferred embodiment, the kit further comprises:

v) a fifth container and a polymerase for amplifying the target DNA contained in the fifth container;

vi) an optional sixth container and reverse transcriptase for reverse transcription, located in the sixth container;

vii) a seventh container and a dNTP for the amplification and/or reverse transcription reaction contained in the seventh container.

In another preferred embodiment, the detection system further comprises reagents used for the Loop Isothermal Amplification (LAMP) reaction.

In another preferred embodiment, the fifth container, the sixth container and the seventh container may be the same container or different containers.

In another preferred embodiment, two, more than two, or all of the first container through the seventh container may be the same container or different containers.

The fourth aspect of the present invention is the development of a method for detecting the presence of target nucleic acid molecules in a sample, which includes the following steps:

(i) obtaining a detection system for target nucleic acid molecules according to the first aspect of the present invention, and the detection system further comprises a sample to be detected; And

(ii) ascertaining whether the nucleic acid probe is cleaved in the Cas12b protein detection system, the cleavage being a trans cleavage of a bypass single-stranded DNA;

however, if the nucleic acid probe is cleaved by the Cas12b protein, this indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas12b protein, this indicates the absence of the target nucleic acid molecule in the sample.

In another preferred embodiment, the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein.

In another preferred embodiment, the sample to be detected comprises a non-amplified sample and an amplified (or amplified nucleic acid) sample.

In another preferred embodiment, the sample to be detected is a sample obtained by amplification.

In another preferred embodiment, the nucleic acid amplification method is selected from the group consisting of: PCR amplification, loop isothermal amplification (LAMP), recombinase polymerase amplification (RPA), ligase chain reaction, branched DNA amplification, nucleic acid sequence based amplification Acids (NASBA), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification, Rolling Ring Amplification, Helicase-Dependent Amplification (HDA), Single Primer Isothermal Amplification (SPIA), Nicking Enzyme Amplification (NEAR), transcription-mediated amplification (TMA) and SMAP2 amplification.

In another preferred embodiment, the PCR comprises a high temperature PCR step, a normal temperature PCR step, or a low temperature PCR step.

In another preferred embodiment, this method is used to detect what is present at the target site - single nucleotide polymorphism (SNP), point mutation, deletion and/or insertion in nucleic acids.

In another preferred embodiment, when the PAM sequence is absent before or after the target site (in the range of -20 nt to +20 nt, preferably in the range of -16 nt to +16 nt), nucleic acid amplification was performed using primers entered using PAM.

In another preferred embodiment, the primer introduced by PAM has the structural formula I from 5' to 3':

P1-P2-P3 (I)

Where,

P1 is the sequence of 5' segments at the 5' end that is complementary or non-complementary to the sequence of the target nucleic acid molecule;

P2 - PAM sequence;

P3 is the sequence of 3' segments at the 3' end that is complementary to the sequence of the target nucleic acid molecule.

In another preferred embodiment, the PAM primer performs specific binding before and after the target nucleic acid molecule.

In another preferred embodiment, P1 is 0-20 nt long.

In another preferred embodiment, P3 has a length of 5 to 20 nt.

In another preferred embodiment, the PAM primer is 16 to 50 nt in length, preferably 20 to 35 nt.

In another preferred embodiment, the complementation comprises complete complementation and partial complementation.

In another preferred embodiment, at least one primer with a PAM sequence is used in amplifying the nucleic acid.

In another preferred embodiment, when the PAM sequence is present before or after the target site (in the range of -20 nt to +20 nt, preferably in the range of -15 nt to +15 nt, more preferably in the range of -10 nt up to +10 nt), primers with or without a PAM sequence can be used and the amplification product contains a PAM sequence.

In another preferred embodiment, the detection in step (ii) comprises a fluorescence detection method.

In another preferred embodiment, a microplate reader or fluorescence spectrophotometer is used in the fluorescence detection method.

The fifth aspect of the present invention is to provide a method for detecting the presence of target nucleic acid molecules in a sample, which comprises the following steps:

(i) obtaining a detection system that contains:

(a) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules;

(c) a nucleic acid probe that is single stranded DNA;

wherein the target nucleic acid molecule is a target DNA;

(d) buffer;

(e1) a polymerase which is used to amplify the target DNA;

(e2) an optional reverse transcriptase which is used for reverse transcription;

(e3) dNTP, which is used for the amplification reaction and/or the reverse transcription reaction; And

(f) sample for testing;

(ii) performing a reverse transcription and/or amplification reaction on the detection system, thereby obtaining a reverse transcription and/or amplified detection system;

(iii) ascertaining whether the nucleic acid probe in the detection system obtained in the previous step is cleaved by the Cas12b protein, the cleavage being a trans cleavage of a bypass single-stranded DNA;

however, if the nucleic acid probe is cleaved by the Cas12b protein, this indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas12b protein, this indicates the absence of the target nucleic acid molecule in the sample.

In another preferred embodiment, in step (ii) the temperature of the detection system is maintained at 50-70°C, preferably 50-65°C, more preferably 55-65°C.

In another preferred embodiment, in step (iii) the temperature of the detection system is maintained at 25-70°C, preferably 48-65°C.

In another preferred embodiment, the nucleic acid amplification method is selected from the group consisting of: PCR amplification, loop isothermal amplification (LAMP), recombinase polymerase amplification (RPA), ligase chain reaction, branched DNA amplification, nucleic acid sequence based amplification Acids (NASBA), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification, Rolling Ring Amplification, Helicase-Dependent Amplification (HDA), Single Primer Isothermal Amplification (SPIA), Nicking Enzyme Amplification (NEAR), transcription-mediated amplification (TMA) and SMAP2 amplification.

In another preferred embodiment, the nucleic acid amplification comprises LAMP amplification.

In another preferred embodiment, in the detection system, the concentration of the nucleic acid in the sample to be tested is from 1×10 ^-11 nM to 1×10 ^-5 nM, preferably from 1×10 ^-9 nM to 1×10 ^-6 nM, more preferably from 1×10 ^-8 nM to 1×10 ^-7 nM.

In another preferred embodiment, the detection system comprises qualitative detection or quantitative detection.

In another preferred embodiment, the assay is an absolute assay (eg, assay combined with digital PCR technology).

In another preferred embodiment, the total time of steps (ii) and (iii) is ≤2 hours, preferably ≤1.5 hours, more preferably ≤1 hour (eg 30-60 minutes).

In another preferred embodiment, the target nucleic acid molecule is a methylated nucleic acid sequence, or the methylated nucleic acid sequence is a nucleic acid sequence obtained after converting unmethylated C to uracil.

In another preferred embodiment, the methylated nucleic acid sequence is treated with bisulfite to convert unmethylated C to uracil.

In another preferred embodiment, the target nucleic acid molecule is a DNA molecule obtained by reverse transcription of a linear or circular RNA molecule, or a DNA molecule obtained by reverse transcription PCR.

In another preferred embodiment, the reverse transcription PCR comprises reverse transcription LAMP.

A sixth aspect of the present invention is the use of a Cas12b protein for preparing a reagent or kit for detecting target nucleic acid molecules based on the single-stranded DNA bypass cleavage activity, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the single-stranded DNA bypass cleavage activity of the Cas12b protein.

In another preferred embodiment, the Cas12b protein is selected from the group consisting of: AacCas12b ( Alicyclobacillus acidoterrestris ), Aac2Cas12b ( Alicyclobacillus acidiphilus ), AkaCas12b ( Alicyclobacillus kakegawensis ), AmaCas12b ( Alicyclobacillus macrosporangiidus ), AheCas12b ( Alicyclobacillus herbarius ), and AcoCas12b (Alicyclobacillus contaminans ).

A seventh aspect of the present invention is to provide a device for detecting the presence of target nucleic acid molecules in a sample, the device comprising:

(a) an amplification reaction module - bypass digestion reaction, which is used to perform nucleic acid amplification reactions and bypass digestion reactions in a digital reaction system in which the bypass reaction is mediated by the Cas12b protein; And

(b) a signal detection module that is used to detect whether a bypass cleavage reaction mediated by the Cas12b protein occurs in each digital reaction system.

In another preferred embodiment, the nucleic acid amplification reaction and the bypass digestion reaction are performed simultaneously.

In another preferred embodiment, the bypass amplification reaction module further comprises a temperature control device for adjusting the temperature of the digital reaction system in the device to a predetermined temperature.

In another preferred embodiment, the predetermined temperature is 50-70°C, preferably 50-65°C, more preferably 55-65°C.

In another preferred embodiment, the predetermined temperature is 25-70°C, preferably 48-65°C.

In another preferred embodiment, the predetermined temperature is essentially the same throughout the amplification reaction and the bypass reaction.

In another preferred embodiment, the predetermined temperature fluctuation range is ±5°C, preferably ±3°C, more preferably ±1°C.

In another preferred embodiment, the nucleic acid amplification is an isothermal amplification or an amplification in which the denaturation-renaturation-extension temperature difference is ≤10°C (preferably ≤5°C, more preferably ≤3°C).

In another preferred embodiment, the nucleic acid amplification comprises LAMP amplification.

In another preferred embodiment, the digital reaction system comprises a digital droplet PCR (ddPCR) reaction system or a digital PCR on chip (cdPCR) reaction system.

In another preferred embodiment, the digital reaction system comprises multiple independent reaction system devices, and each reaction system device contains:

(a) a Cas12b protein, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

(b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules;

(c) a nucleic acid probe that is single stranded DNA;

wherein the target nucleic acid molecule is a target DNA;

(d) buffer;

(e1) a polymerase that is used to amplify the target DNA;

(e2) an optional reverse transcriptase which is used for reverse transcription;

(e3) dNTP, which is used for the amplification reaction and/or the reverse transcription reaction; And

(f) sample for testing;

however, due to dilution processing (ie, digital processing), each independent reaction system contains either 1 copy of the nucleic acid from the sample to be tested or 0 copies of the nucleic acid from the sample to be tested.

In another preferred embodiment, each device of the reaction system is the same except for 1 or 0 copies of the nucleic acid from the sample to be tested.

In another preferred embodiment, the device of the reaction system is a drop.

In another preferred embodiment, the reaction system device is a microreaction system located in the micropores of the chip.

In another preferred embodiment, the device further comprises a sample loading module and/or a control module.

In another preferred embodiment, the signal detection module comprises a visualization module.

In another preferred embodiment, the imaging module comprises a fluorescent detection device.

In another preferred embodiment, the fluorescence detection device irradiates the digital reaction system to excite the reaction system to generate a fluorescent signal and converts the fluorescent signal into a digital signal, which is preferably sent to a control module.

In another preferred embodiment, the control module performs an arithmetic analysis based on the number of fluorescent signals (in the preferred embodiment, digital signals converted from fluorescent signals) and the total number of devices in the reaction system, thus obtaining a target nucleic acid quantitation result (e.g., concentration or number of copies).

In another preferred embodiment, the device is a digital PCR detection device.

It should be understood that within the scope of the present invention, the various technical features of the present invention above and the various technical features described below (eg, in the embodiments) may be combined with each other to create a new or preferred technical solution. Due to space limitations, it is not repeated in this document.

Description of figures

In FIG. 1 shows the fluorescence intensity generated by trans- cleavage of probes with various concentrations of Cas12b and a single guide RNA using double-stranded DNA and single-stranded DNA as the target DNA, respectively.

In FIG. 2 shows the effect of different bases in the PAM sequence on the intensity of fluorescent detection of trans cleavage using double-stranded DNA and single-stranded DNA as target DNA, respectively.

In FIG. 3 shows the rate of trans cleavage by the Cas12b protein using double-stranded DNA or single-stranded DNA as a target.

In FIG. 4 shows the fluorescence intensity of trans- cleavage by the Cas12b protein using double-stranded DNA or single-stranded DNA as a target under various temperature conditions.

In FIG. 5 shows the detection sensitivity of the Cas12b protein (ie, trans- cleavage fluorescence intensity) using double-stranded DNA or single-stranded DNA as the target during gradient dilution of the target sequence.

In FIG. 6 shows the detection sensitivity of the Cas12b protein in combination with the LAMP amplification reaction (ie HOLMES v2.0 method) on the target.

In FIG. Figure 2 shows the effect of single-base mutations at positions 1–12 of the target sequence of single-stranded DNA on the fluorescence intensity of trans- cleavage by the Cas12b protein using single guide RNAs with different lengths of the guide sequence.

In FIG. Figure 8 shows the effect of single base mutations at positions 1-16 of the single-stranded DNA target sequence on the fluorescence intensity of trans-cleavage by the Cas12b protein using a single guide RNA with a 16 nt guide sequence.

In FIG. Figure 9 shows the effect of single base mutations at any of the other three bases at positions 1-16 of the target single-stranded DNA sequence on the fluorescence intensity of trans-cleavage by the Cas12b protein using a single guide RNA with a 16 nt guide sequence.

In FIG. Figure 10 shows the effect of single base mutations at positions 8–16 of three different single-stranded DNA target sequences (rs5082, rs1467558, and rs2952768) on the fluorescence intensity of trans-cleavage by the Cas12b protein using a single guide RNA with a 16 nt guide sequence.

In FIG. Figure 11 shows the effect of single base mutations in any of positions 1–16 of the double-stranded DNA target sequence on the fluorescence intensity of trans-cleavage by the Cas12b protein when using single guide RNAs with different lengths of the guide sequence.

In FIG. 12 shows one-step LAMP reaction and detection by Cas12b protein at 50°C, 55°C, 60°C and 65°C.

In FIG. 13 shows the results of the HOLMES v2.0 tests (LAMP in combination with the Cas12b protein) for the E. coli gyrB site to detect the presence of E. coli and the detection sensitivity in the gradient dilution test. (After the coating test, the number of 0.005 OD E. coli was about 7000)

In FIG. 14 shows the results of the HOLMES v2.0 tests (LAMP in combination with the Cas12b protein) for the sex-determining region of the Y chromosome using saliva samples for sex determination (the DNMT1-3 locus is the gene locus on the chromosome that was used as a positive control) .

In FIG. 15 shows the detection of the SNP site (rs5082) by asymmetric or symmetrical PCR amplification in combination with the Cas12b protein. It can be seen that since there is no PAM sequence near the rs5082 site, asymmetric PCR, which produces single-stranded DNA, can recognize SNP sites more clearly.

In FIG. 16 shows the detection of the SNP site (rs5082) by LAMP amplification in combination with the Cas12b protein, with primers designed and the PAM sequence introduced. It can be seen that since there is no PAM sequence near the rs5082 site, the LAMP amplification method used with PAM (with the PAM sequence in the primer) allows better recognition of SNP sites.

In FIG. 17 shows detection of JEV (Japanese encephalitis virus) RNA by HOLMES v2.0 method (LAMP in combination with Cas12b protein).

In FIG. 18 shows one-step trace DNA quantitation by the HOLMES v2.0 method (LAMP in combination with Cas12b protein). With template DNA diluted at various concentrations, the one-step LAMP-Cas12b method was used to detect real-time fluorescence value in a fluorometer (reaction at 55°C).

In FIG. 19 shows an exemplary linear trend using the time when the fluorescence value reaches 600,000 as the y-axis (using the data in FIG. 18) and using the absolute concentration lg as the x-axis. This equation can be used to quantify the target sequence.

In FIG. 20 shows the absolute quantitation of nucleic acid samples using the digital HOLMES method (LAMP in combination with Cas12b protein and a transparency chip).

In FIG. 21 shows a schematic for detection of target DNA methylation by the HOLMESv2 method. After bisulfite conversion and PCR amplification, base C in the unmethylated CpG site is converted to T. By constructing a single RNA guide sequence that exactly matches the methylated target site, the HOLMESv2 reaction (namely, adding DNA labeled with a fluorescent group at one end and a quench group at the other end) was used to recognize the content of methylated sites in the system. Similarly, one can also design a single RNA guide sequence that exactly matches the unmethylated target site and use the HOLMESv2 reaction to recognize the content of unmethylated sites in the system, and then the ratio of methylated sites can be calculated.

In FIG. 22 shows the experimental results of detection of target DNA methylation using single guide RNAs with different sites and different lengths. Target-C means the test result at position C (representing methylation); while target-T means the test result at the T position (representing the unmethylated base before the bisulfite conversion). If a single guide RNA sequence has a high signal value when targeting C-target and a low signal value when targeting T-target, the single guide RNA sequence can be used to recognize if the site has the ability to modify methylation.

In FIG. 23 shows a scheme for detecting target DNA methylation using a single guide RNA at m3-C12-17. In the left picture, you can see that the sequence of a single guide RNA is exactly the same as the target sequence; while the right picture shows that after bisulfite conversion and PCR amplification of the unmethylated site, the M3 site is converted to T, resulting in a mismatch between the single guide RNA sequence and the target sequence.

In FIG. 24 shows the results of testing the level of methylation of the M3 CpG site ( COL1A2 ) in 4 cell lines. The 0%, 10%, 30% and 50% results are test results for the calibration curve. 293T, SW480, NCI-N87 and MCF-7 are the actual test values at this location in 4 cell lines. From left to right, 3 independent replicate experiments are shown, and 3 replicate tests were performed for each experiment.

In FIG. 25 shows a calibration curve for the level of methylation at the M3 CpG site ( COL1A2 ). The calibration curve diagram is made based on the result of checking the calibration curve in FIG. 5.

In FIG. 26 shows the results of testing the methylation rate of the M3 CpG site ( COL1A2 ) in four cell lines (293T, SW480, NCI-N87 and MCF-7). As can be seen from the figure, the result of testing with the HOLMESv2 system is closest to the result of NGS sequencing (high throughput sequencing).

In FIG. 27 shows the results of cRNA detection using the HOLMESv2 method. The top figure shows a diagram depicting RNA detection using the HOLMESv2 method and target site selection. The lower left figure shows a diagram depicting target GAPDH detection using the HOLMESv2 method. The lower right figure shows a diagram depicting target CDR1as detection using the HOLMESv2 method. Target site means a target site. NTC is an experimental control group that uses sterile water as a sample. A target is an experimental group that uses an RNA sample as a sample. The fluorescence values in the lower left and lower right plots are the fluorescence values after subtracting the experimental background fluorescence. Both reaction groups and the NTC target group were amplified by the LAMP reaction, and the amplified products were used to test for the Cas12b reaction; and background fluorescence was the fluorescence value of the Cas12b reaction system tested by adding sterile water directly.

Detailed description

Through extensive and thorough research, the inventors of the present invention pioneered a technical solution for the detection of a target nucleic acid by studying the cleavage properties of Cas12b enzymes. The experimental results show that the solution of the present invention allows fast, highly sensitive and accurate detection of nucleic acid and that it can be applied in various fields, for example, rapid sex identification using saliva samples, detection of E. coli infection, rapid identifying SNP genotypes, detecting virus RNA, and determining the concentration of trace DNA in a sample. On this basis, the present invention was created.

Terms

The term "guide RNA" or "gRNA" or "sgRNA" (single guide RNA) means an RNA that directs a Cas protein (eg Cas12b protein) to specifically bind to a target DNA sequence.

The term "CRISPR" refers to short, regular-spaced, palindromic cluster repeats that represent the immune systems of many prokaryotes.

The term "Cas protein" means a CRISPR-associated protein, which is a protein associated with the CRISPR system.

The term "Cas12a" (formerly "Cpf1") means crRNA-dependent endonuclease, which is a type V-A enzyme in the classification of the CRISPR system.

The terms "Cas12b", "C2c1" are used interchangeably and refer to sgRNA-dependent endonuclease, which is a type V-B enzyme in the classification of the CRISPR system.

The term “LAMP” means a loop isothermal amplification method and a thermostatic nucleic acid amplification method suitable for gene diagnostics.

The term "PAM" means a motif adjacent to the protospacer, which is necessary for cleavage of double-stranded DNA by the Cas12b protein, and PAM AacCas12b is the TTN sequence.

The term "PCR" means "polymerase chain reaction", a technique used to amplify a large number of DNA fragments of interest.

HOLMES v2.0 stands for One-Hour Low Cost Multipurpose High Efficiency System Version 2.0, which is a nucleic acid detection method based on the Cas12b protein.

Detection method

The present invention provides a method for detecting target nucleic acid molecules, which includes adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer to a reaction system containing the target nucleic acid molecules, and then determining the fluorescence intensity.

The present invention provides a method for rapidly detecting target nucleic acid molecules with high specificity. After the target DNA (single-stranded or double-stranded), the single-stranded guide RNA and the Cas12b protein form a ternary complex, this complex cleaves the single-stranded DNA molecules in the system.

In the present method, a target DNA (DNA sequence to be detected) is targeted by an engineered single guide RNA; and single guide RNA and Cas12b protein are added to the detection system. When a target DNA (single-stranded DNA or double-stranded DNA) is present, the Cas12b protein, single guide RNA and target DNA form a ternary complex, and this complex cleaves a nucleic acid probe labeled with a fluorescent signal using its bypass cleaving activity, thus emitting fluorescence.

The representative nucleic acid probe is a single-stranded DNA with a fluorescent group and a quenching group attached to both ends, so after the probe is cleaved, the fluorescent group can emit light.

In the present invention, by detecting fluorescence, it can be known whether the target DNA molecule is contained in the system to be detected.

In the present invention, the suitable Cas12b protein is Cas12b, and the preferred Cas12b protein is: AacCas12b ( Alicyclobacillus acidoterrestris ), Aac2Cas12b ( Alicyclobacillus acidiphilus ), AkaCas12b ( Alicyclobacillus kakegawensis ), AmaCas12b ( Alicyclobacillus macrosporangiidus ), AheCas12b ( Ali cyclobacillus herbarius ) or AcoCas12b (Alicyclobacillus contaminans ) .

The target nucleic acid molecules to be detected in the reaction system containing the target nucleic acid molecules to be detected may be non-amplified or obtained after amplification and/or after reverse transcription amplification.

The detection method of the present invention can detect nucleic acid molecules of various kinds, for example, nucleic acid molecules of mammals, plants, microorganisms, and viruses. The method of the present invention is particularly suitable for the detection of pathogens, gene mutations, or specific DNA or RNA targets.

The method of the present invention makes it possible to quickly ascertain whether a specific DNA sequence is contained in a sample. In addition, when combined with an amplification method (eg, LAMP, PCR, asymmetric PCR, RPA, etc.), the sensitivity of the detection method can be greatly improved.

In a preferred embodiment, Cas12b bypass digestion can be combined with nucleic acid amplification (eg, isothermal amplification, LAMP amplification) and other methods (eg, digital PCR).

In a preferred embodiment of the present invention, when Cas12b bypass detection is combined with nucleic acid amplification, detection sensitivity can be increased to a concentration of 10 ⁻⁸ nM or lower.

In addition, when Cas12b bypass detection is combined with digital PCR, detection sensitivity can be increased to 1 copy per reaction system (eg, microdroplet), which meets almost all detection sensitivity needs.

Method for Nucleic Acid Detection Based on Cas12b Protein and HOLMES v2.0

Creation of nucleic acid detection methods

Using the characteristics of the Cas12b protein, the present invention developed a method for the specific detection of nucleic acid molecules, which is called HOLMES (one-hour low-cost multi-purpose high-performance system version 2.0). As the name suggests, this is a fast (1 hour), low cost, versatile, efficient and easy way to test.

In the entire reaction system, it can be divided into two large stages. One step is the amplification of the template nucleic acid, and the other is the specific detection of the nucleic acid using the Cas12b protein. Alternatively, the two steps may be combined into one step.

In the present invention, in a preferred embodiment, the LAMP method or asymmetric PCR is used to amplify nucleic acids. But in practice, any amplification method can be combined with second step nucleic acid detection, such as RPA isothermal amplification method or the like.

The parent nucleic acid is not limited to double-stranded DNA, and may be single-stranded DNA or RNA, and thus this method is applicable to various types of nucleic acid molecules.

In the nucleic acid detection step, three components play an important role for the experiment, namely the Cas12b protein, the single guide RNA, and the nucleic acid probe. In addition to AacCas12b mentioned in the examples, other Cas12b proteins are equally suitable for the present method. In addition, other types of Cas proteins (eg, Cas12c protein) are also within the scope of the present invention.

As for a single guide RNA as a guide, after artificial modification and other modifications, it will be more stable in the system. In the choice of nucleic acid probes in the present invention, short single stranded DNA labeled with HEX and BHQ1 (also labeled with FAM and Eclipse) is selected, and any other method with a detectable label is theoretically applicable, provided that the nucleic acid probe after cleavage can create detectable difference. Alternatively, a nucleic acid probe can also be designed to emit fluorescence after binding to a compound to determine if the probe has been cleaved or not.

For ease of understanding, the various features of the Cas12b nucleic acid detection method (including the preferred HOLMES v2.0 method) of the present invention are described below.

Identification of the trans- cleaving activity of Cas12b: Although the proteins Cas12a and Cas12b are completely different in sequence, the experimental results obtained by the present inventors show that the Cas12b protein also has a trans -cleaving activity.

Initially, the bypass DNA was designed as a fluorescent probe consisting of a random 12 nt sequence, and the fluorescent group was labeled at the 5' end, and the BHQ1 quench group was labeled at the 3' end (HEX-N12-BHQ1). When the system contains a target DNA fragment (double-stranded DNA or single-stranded DNA), a ternary complex is formed from the target DNA, a single guide RNA, and the Cas12b protein. At this point, the probe splits and the fluorescence emitted by the HEX fluorescent group can be detected with a fluorescent detector (535 nm excitation light, 556 nm emission light).

As shown in FIG. 1, when the concentration of Cas12b and single guide RNA was 250 nM or 500 nM, and double-stranded DNA and single-stranded DNA as targets showed relatively high fluorescence intensity. When the concentration of Cas12b and the single guide RNA was reduced to 50 nM and 100 nM, the single stranded DNA as the target sequence still showed high fluorescence intensity.

Characteristics of PAM trans cleavage of Cas12b: Studies have shown that during cis cleavage of double-stranded DNA by the Cas12b protein, a sequence called PAM is required. In other words, only the 5' end followed by the 5'-TTN-3' can be truncated by the Cas12b protein. In order to know if PAM is required for the Cas12b target sequence during trans cleavage, the present inventors designed various sequences (including TTC, TAC, ATC, AAC, GGC and CCC) at the location of PAM to find out if PAM is required during trans splits.

As shown in FIG. 2, when double-stranded DNA is used as a target, trans- cleavage with the Cas12b protein is more sensitive to the PAM sequence. In other words, increased trans- cleavage fluorescence intensity can be obtained when the TTC sequence is used (the fluorescence intensity generated after changing the sequence to TAC is slightly lower than the fluorescence intensity with the TTC sequence). And when single-stranded DNA is used as a target, it is insensitive to the PAM sequence and always shows a high fluorescence intensity when trans- cleaved.

Trans cleavage rate by Cas12b protein: When single-stranded DNA and double-stranded DNA are used as target DNA, respectively, the trans cleavage rate by Cas12b protein is measured.

In FIG. Figure 3 shows that when single-stranded DNA is used as a substrate, the rate of trans cleavage by Cas12b is very high, approaching its highest value after only 6 minutes. When double-stranded DNA is used as a substrate, the cleavage rate is relatively slow and a gradual increase occurs. This means that in some cases where rapid detection is required (eg, ≤10 minutes), single-stranded DNA must be used as the substrate for Cas12 bypass cleavage.

Applicable temperature range for trans cleavage with Cas12b: Cas12b has a very wide temperature range for trans cleavage, whether the target DNA is single-stranded or double-stranded. A temperature of 45-65°C has a good response value, especially given that the temperature range for single-stranded DNA target is wider, namely 25-70°C (see Fig. 4).

Cas12b Detection Sensitivity : The present inventors tested the detection sensitivity of Cas12b by diluting the concentration of double-stranded target DNA or single-stranded target DNA. In a preferred embodiment, a single-stranded DNA target (DNMT1-3(TTC PAM)-R) or a double-stranded DNA target (obtained by annealing two oligonucleotides DNMT1-3(TTC PAM)-F and DNMT1-3(TTC PAM)-R) was diluted up to concentrations of 100 nM, 10 nM, 1 nM, ... up to 10 ^-4 nM.

The results show that when the DNA concentration is 1 nM, the target molecule can still be detected by Cas12b (FIG. 5).

HOLMES v2. Sensitivity Test: In one example, when Cas12b and the LAMP reaction were combined (i.e. HOLMES v2.0), the detection sensitivity could reach 10 ^-8 nM, which significantly increased the detection sensitivity of the target nucleic acid sequence (Fig. .6).

SNP Versus Single Stranded DNA Target Assay : The method of the present invention is very suitable for the detection of nucleic acid mutations, including single nucleotide polymorphisms, especially single nucleotide polymorphisms in single stranded DNA targets.

Detection of single-stranded DNA target SNPs showed that when the length of the single-stranded guide RNA guide sequence was 18 nt or 20 nt, the base mutation of positions 1-12 had little effect on the fluorescent signal produced by trans cleavage. When the guide length of the single guide RNA was 14 nt or 15 nt, the fluorescence values, including the wild-type control, were very low. When the guide length of the single guide RNA was 16 nt, after mutation of positions 10-12, the fluorescence value decreased significantly compared to the control (Fig. 7).

Upon further investigation, it was found that when the length of the single guide RNA guide sequence was 16 nt, mutation of the base of positions 10–16 had an obvious effect on the fluorescence value, especially given that the fluorescence value was almost undetectable after the mutation of positions 10–14 (Fig. 8).

In addition, when different positions of the single-stranded target DNA were mutated into different types of bases, this did not significantly affect the fluorescence value. However, only a mutation in the G position 9 significantly changed the fluorescence value (Fig. 9).

In addition, the present inventors also tested three other target sequences. Although different sequences had different fluorescence changes as a result of mutation of a specific site, the position resulting in a significant change could still be found for each sequence among positions 8-16, for example, positions 8, 10, 11 and 12 in rs5082, positions 11 in rs1467558, positions 8–15 in rs2952768 (Fig. 10).

SNP Versus Double Stranded DNA Target Assay : The method of the present invention is suitable for the detection of nucleic acid mutations, including single nucleotide polymorphisms in double stranded DNA targets.

Detection of the double-stranded DNA target SNP showed that the fluorescent signal produced by trans- cleavage due to a single base mutation differs from the fluorescent signal of single-stranded DNA. For example, a single guide RNA with a guide sequence of 18–20 nt has varying degrees of sensitivity to base mutations at positions 10–16 (although the fluorescent signal also decreases to varying degrees after base mutations at positions 1 and 3) (Fig. eleven). Typically, these single guide RNAs are most sensitive to positions 10 and 16.

One-step detection at different temperatures : The present invention also proposes a one-step detection that combines nucleic acid amplification and Cas12b.

For one-step detection, it is preferable to combine LAMP amplification with Cas12b protein detection. In the present invention, trans cleavage by the Cas12b protein was tested under various temperature conditions (50°C, 55°C, 60°C and 65°C, respectively). As shown in FIG. 12, one-step detection can be achieved at 50 and 55°C. The fluorescence value increases faster especially at 55°C.

Target RNA Testing: The method of the present invention is suitable not only for target DNA detection, but also for target RNA detection. In the present invention, RNA can be reverse transcribed into DNA form, after which the target sequence can be detected.

The preferred method is to add reverse transcriptase to the reaction system to obtain reverse transcription of the RNA. Then, for example, Bst 2.0 DNA polymerase is used for amplification, and Cas12b protein is used to obtain a trans cleavage. Another method is to directly use the Bst 3.0 DNA polymerase (this enzyme can amplify messenger RNA to produce DNA) to reverse transcription and RNA amplification directly (FIG. 17).

Quantification of Trace DNA Template The method of the present invention can be used for quantitation.

In one example, a one-step method based on LAMP amplification and Cas12b was used at 55°C, thus performing trace DNA quantitation. When the matrices were diluted to different concentrations, the trans- cleavage fluorescence value was determined in real time. Thereafter, a calibration curve was generated and the concentration of the target template sequence can be obtained by calculation (FIGS. 18 and 19).

Another quantification method is to combine the Cas12b protein detection method of the present invention with a digital PCR method (see "HOLMES Digital Method" below).

Design of primers and single guide RNA

Based on the idea of the present invention, those skilled in the art can synthesize appropriate primers and/or single guide RNAs based on practical needs for detecting the Cas12b nucleic acid of the present invention. In the present invention, some preferred primer and single guide RNA designs may be referred to as the following sentences.

With regard to primers, design may refer to the following.

Situation 1. If the LAMP method is chosen for the previous amplification reaction, conventional primer design methods can be used (http://primerexplorer.jp/elamp4.0.0/index.html). It is recommended to add LoopF and LoopB primers between the F2/F3 and B2/B3 primer regions, i.e. 6 pairs of primers.

Situation 2: If the PCR method is chosen for the previous amplification reaction, several primers must be prepared to test for the most suitable combination to obtain single-stranded DNA.

Situation 3. For SNP discovery, if a suitable PAM site is near the site, then the solution for Situation 1 is used; if there is no suitable PAM site, then the PAM site must be primed in order for the product to create the PAM site. Usually the SNP site is 8-16 bases away from PAM, but the specific target sequence needs to be tested.

Situation 4: If it is difficult to find suitable primers for LAMP amplification near the SNP site or it is difficult to introduce a suitable PAM, methods such as asymmetric PCR amplification can be used to amplify single-stranded DNA.

With respect to the construction of a single guide RNA, the following situations can be considered.

Situation 1. For the detection of target genes, it is usually advisable to choose a complementary paired single guide RNA 20 bp. in the case of a target sequence with a PAM sequence.

Situation 2: For SNP detection, if double-stranded amplification such as LAMP is used in the amplification step, the position of the SNP site must first be checked. Single guide RNAs with a guide sequence length of 18 nt are tested first for positions 10–16.

Situation 3: For SNP detection, if single strand amplification, such as asymmetric PCR, is used in the amplification step, the position of the SNP site must first be checked. Single guide RNAs with a guide sequence length of 16 nt are tested primarily for positions 10–16, especially for positions 10–12.

Digital way HOLMES

The present invention also provides a method that combines the HOLMES method of the present invention with digital PCR (dPCR), referred to as "Digital HOLMES method" for short.

Digital PCR is a technique for the absolute quantification of nucleic acid molecules that has developed rapidly in recent years. At present, two types are mainly applied, digital drop PCR (ddPCR) and digital PCR on chips (cdPCR). In other words, prior to conventional PCR amplification, the sample is converted into droplets or added to a chip, and the reaction system containing nucleic acid molecules is separated into thousands of reaction systems at the nanoliter level. Compared to traditional PCR, digital PCR additionally has a pre-processing step and later a fluorescence detection step. At present, detection by digital PCR still requires the use of a traditional PCR device to perform the amplification step, and the PCR reaction conditions are more stringent and take longer (about 3-4 hours).

When using the HOLMES method in combination with the currently available fluorescence reader for digital PCR on chips (i.e. digital HOLMES method), absolute quantitation can only be performed at a constant temperature.

In addition, when the LAMP method or other methods are used for amplification in the HOLMES digital method, the amplification can be carried out over a wide temperature range (50-70°C or about 50-55°C), without the need for precise temperature control, as is the case when using conventional PCR devices. Because Cas12b also has a bypass cleavage activity in this temperature range, it can not only integrate effectively, but also perform amplification and bypass cleavage simultaneously.

The present invention also provides a device for digital detection HOLMES, in particular the device according to the seventh aspect of the present invention.

Therefore, the HOLMES digital method can be used to integrate pre-processing, amplification and detection in one instrument, which can not only reduce human error during operation, but also speed up the detection process and reduce instrument maintenance costs.

Sets

The present invention also provides a kit containing a guide RNA, a 12b protein and a nucleic acid probe. In addition, the kit of the present invention may also contain other reagents such as buffers, reagents required for amplification, reagents required for reverse transcription, or a combination thereof.

Typically, a kit of the present invention contains:

i) a first container and a Cas12b protein contained in the first container, wherein the Cas12b protein is a Cas12b or Cas protein having an activity similar to the bypass cleavage activity of the single-stranded DNA of the Cas12b protein;

ii) an optional second container and a second guide RNA contained in the second container, the second guide RNA directing the Cas12b protein for specific binding to target nucleic acid molecules;

iii) a third container and a nucleic acid probe (preferably a fluorescent probe) contained in the third container;

iv) an optional fourth container and a buffer located in the fourth container;

wherein the target nucleic acid molecule is a target DNA.

In addition, the kit of the present invention may further comprise:

v) a fifth container and a polymerase for amplifying the target DNA contained in the fifth container;

vi) an optional sixth container and reverse transcriptase for reverse transcription, located in the sixth container;

vii) a seventh container and a dNTP for the amplification and/or reverse transcription reaction contained in the seventh container.

In the present invention, one, several or all of the containers may be the same container or different containers.

The main advantages of the present invention include:

Using the characteristics of the Cas12b protein, the inventors of the present invention have developed a method for the specific detection of nucleic acid molecules, which is called HOLMES (one-hour low-cost multipurpose high-performance system version 2.0). It is characterized as fast (1 hour), low cost, versatile, efficient and easy way to test. This method can be used in the field of rapid pathogen detection, SNP detection, and the like. Compared with the first generation HOLMES method based on Cas12a, the second generation HOLMES method based on high temperature Cas12b enzyme has more advantages.

(1) Fast: When the testing conditions are ready, it only takes about 1 hour from the time the sample is received to the time the result is received.

(2) Low cost: The experiment does not require any special materials or enzymes, and the amount of materials and reagents used is small. It can be used for testing and trace analysis.

(3) Efficiency: The method of the present invention is extremely sensitive and can detect DNA at a concentration of 10 aM.

(4) Versatility: It can detect various nucleic acid samples, including DNA samples and RNA samples.

(5) Simplicity: Does not require any special complicated steps. If the kit is ready and the program is set, you just need to add a sample or perform some similar step.

(6) One-step reaction: The amplification reaction and the detection reaction can be carried out at the same time, making this test a one-step test.

(7) Isothermal: Because both LAMP amplification and Cas12b protein detection are performed at a constant temperature, the instrument requirements are lower.

(8) Wide reaction temperature range: Cas12b can be used for detection at 45-65°C, so the instrument requirement is lower.

(9) Two enzymes can carry out amplification and detection of DNA or RNA: Since Bst 3.0 DNA polymerase has the activity of DNA polymerase using DNA or RNA as a template, only two enzymes, Bst 3.0 and Cas12b, are required to carry out amplification and detection DNA or RNA, which reduces production costs.

(10) High Sensitivity SNP Detection: With proper site design and a single guide RNA, single base differences can be recognized with high sensitivity.

(11) Quantitative detection is possible: Real-time fluorescence detection by the HOLMES one-step method or combined with the chip and detection system of the digital PCR instrument allows quantitative detection of samples.

The following description of the invention is given together with specific embodiments. It should be understood that these examples are not intended to limit the scope of the invention. The experimental methods in the following examples, where no specific conditions are specified, are generally performed under conventional conditions, such as those described in Sambrook et al. "Molecular Cloning": A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or under the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

In this application, unless otherwise indicated, the Cas12b trans -cleaving protein system contains: 250 nM single guide RNA and 250 nM Cas12b protein and reaction conditions: 48 °C for 30 minutes.

materials

1. The RNase inhibitor was purchased from TaKaRa. KOD FX high fidelity DNA polymerase was purchased from ToYoBo. Primers (oligonucleotides) were synthesized by Shanghai Biotech. T7 RNA polymerase was purchased from Thermo. An RNA purification and isolation kit (RNA Clean & Concentrator ^TM -5) was purchased from Zymo Research. The Wizard® SV Gel Purification and PCR System was purchased from Promega. The medium (eg tryptone, yeast extract, etc.) was purchased from OXOID.

2. Medium formula: liquid LB (1% tryptone, 0.5% yeast extract and 1% NaCl). When formulating solid LB, only 2% agar needs to be added to liquid LB.

Example 1

Detection of double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) targets using the Cas12b protein

Single-stranded DNA or double-stranded DNA (T1 target) was chosen as the target sequence to test the response values of Cas12b protein and single guide RNA at various concentrations.

1. Preparation of guide RNA (single guide RNA)

First, the pUC18-sgRNA-T1 plasmid was constructed with pUC18 as the plasmid backbone. In this plasmid, the T7 promoter and template DNA sequence for transcription of the single guide RNA were introduced with pUC18. (Note: A single guide RNA transcribed from this template targets a sequence referred to as T1 in this study.) In this preparation, plasmid pUC18 was used as a template, and pUC18-1-F and pUC18-1-R were used as primers, and a first round of PCR was performed. T4 DNA ligase was then used to join the PCR products and the reaction product was transformed into DH10b. After sequencing, the correct clone was obtained and named pUC18-sgRNA-T1-pre. Then the plasmid pUC18-sgRNA-T1-pre was used as a template, and pUC18-2-F and pUC18-2-R were used as primers, and a second round of PCR was performed. The PCR products were connected in the same way and transformed. Finally, the correct sequenced pUC18-sgRNA-T1 plasmid was obtained.

Then the pUC18-sgRNA-T1 plasmid was used as a template, and T7-crRNA-F and sgRNA-T1-R were used as primers, and the DNA template required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA (referred to as sgRNA-T1) was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Finally, DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. Preparation of target DNA

(1) If the target DNA was single-stranded, an oligonucleotide target DNA (T1-R) was directly synthesized, which contains 20 bp. a target sequence (T1) recognized by a single guide RNA.

(2) If the target DNA was double-stranded, two complementary oligonucleotides (T1-F; T1-R) were directly synthesized, which contain 20 bp. a target sequence (T1) recognized by a single guide RNA. Two oligonucleotides were annealed and thus a double-stranded DNA target was obtained. More specifically, paired oligonucleotides (2 μM) were mixed in 1×PCR buffer (Transgen Biotech) in a total volume of 20 μl. After that, an annealing procedure was performed: first, denaturation was carried out at 95°C for 5 minutes, then cooling from 95 to 20°C at a cooling rate of 1°C per minute, using a thermal cycler.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to a suitable concentration and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To the 20 µl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (the final concentration of both components was 50, 100, 250, or 500 nM), a fluorescent probe, and a quench probe (HEX- N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. After that, it was inactivated by heating at 98°C for 5 minutes.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

The results are shown in FIG. 2. When the concentration of Cas12b and single guide RNA was 250 or 500 nM, both double-stranded DNA and single-stranded DNA as targets showed relatively high fluorescence intensity. When the concentration of Cas12b and the single guide RNA was reduced to 50 and 100 nM, the single stranded DNA as the target sequence still showed high fluorescence intensity.

Example 2

Response sensitivity test using HOLMES v2.0 method (LAMP combined with Cas12b)

By detecting the excited fluorescence intensity of the fluorescent probe (HEX-N12-BHQ1), the target DNA concentration required for the bypass cleavage activity of single-stranded DNA of the Cas12b protein, in other words, the sensitivity of the bypass reaction of single-stranded DNA cleavage by the Cas12b protein, was determined.

1. Preparation of single guide RNA

First, the previously constructed plasmid pUC18-sgRNA-T1 was used as a template, and primers sgRNA-DNMT1-3-F and sgRNA-DNMT1-3-R were designed. The 20 bases targeting the T1 DNA target in the single guide RNA were replaced by a single guide RNA targeting DNMT1-3 by PCR, resulting in another plasmid pUC18-sgRNA-DNMT1-3.

Then the plasmid pUC18-sgRNA-DNMT1-3 was used as a template, and T7-crRNA-F and ZLsgRNA-DNMT1-3-R were used as primers, and the DNA template required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA (sgRNA-DNMT1-3) was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Finally, DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. Plasmid DNA was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. Preparation of target DNA

For the target DNA, the first situation was the addition of the Cas12b protein to the reaction system directly, without amplification. The description of the method is given below:

(1) If the target DNA was single stranded, the 50 bp oligonucleotide (DNMT1-3) was synthesized directly to be used as a target DNA, which contains 20 bp. a target sequence (DNMT1-3) recognized by a single guide RNA.

(2) If the target DNA was double stranded, two complementary 50 bp oligonucleotides were directly synthesized. (DNMT1-3(TTC PAM)-F; DNMT1-3(TTC PAM)-R), which contain 20 p. a target sequence (DNMT1-3) recognized by a single guide RNA. Two oligonucleotides were annealed and thus a double-stranded DNA target was obtained. More specifically, paired oligonucleotides (2 μM) were annealed in 1×PCR buffer (Transgen Biotech) in a total volume of 20 μl. After that, an annealing procedure was performed: first, denaturation was carried out at 95°C for 5 minutes, then cooling from 95 to 20°C at a cooling rate of 1°C per minute, using a thermal cycler.

(3) Single-stranded or double-stranded DNA target was diluted to 2 μM, 0.2 μM, 0.02 μM, 0.002 μM and 0.0002 μM for use.

The second situation was that the fragment containing the target sequence (DNMT1-3) was introduced into a plasmid vector and amplified by the LAMP reaction.

(1) The pEasy-Blunt Zero Cloning Kit from Transgen was used to introduce the fragment containing the target sequence (DNMT1-3) into the pEasy-Blunt Zero Cloning Vector, and the correct clone was obtained after sequencing verification.

(2) LAMP amplification reaction

The above plasmid was used as a template for performing LAMP amplification. The matrix was added at 0.1 nM, 0.1 nM, and 10-fold dilution concentrations to 10 ^-11 nM, respectively. The total volume of each reaction system was 25 μl. 1.6 µM LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 µM LAMP-DNM-F3 and LAMP-DNM-B3, and 0.4 µM LAMP-DNM-LoopF and LAMP-DNM-LoopB were used in as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: In 20 µl of the reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (final concentration of both components was 250 nM), 1 µl of target DNA or 1 µl of LAMP product, fluorescent probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. After that, it was inactivated by heating at 98°C for 5 minutes.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

In FIG. 5 shows that Cas12b can detect single or double stranded DNA at a concentration of 1 nM. In combination with LAMP amplification of Cas12b (ie HOLMES v2.0 method) can detect DNA at 10 ^-8 nM (FIG. 6).

Example 3 Target Single Base Mutation Detection Using Cas12b Protein

Single-stranded DNA or double-stranded DNA was used as a target sequence to test for a change in the fluorescent trans- cleavage signal of the Cas12b protein in the presence of a single-base mutation in the target DNA, thus detecting single-base mutations.

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as a template, T7-crRNA-F was used as a forward primer, and oligonucleotides containing guide sequences complementary to different targets were used as reverse primers. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template, and full-length single guide RNA (with a guide sequence of 20 nt) or truncated single guide RNA (with a guide sequence of less than 20 nt) was synthesized using the T7 High Yield Transcription Kit (Thermo) . The reaction proceeded at night (12–16 h) at a temperature of 37°C.

DNase I was then added to the transcription system (2 µl DNase I (5 U/µl) per 50 µl transcription system), incubated in a water bath at 37°C for 30 minutes. The template DNA was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. Preparation of target DNA

(1) If the target DNA was single stranded, the 50 bp oligonucleotide synthesized directly to be used as a target DNA that contains a target sequence recognized by a single guide RNA.

(2) If the target DNA was double-stranded, two complementary 50 bp oligonucleotides were directly synthesized that contain the target sequence recognized by the single guide RNA. Two oligonucleotides were annealed and thus a double-stranded DNA target was obtained. More specifically, paired oligonucleotides (1 μM) were annealed in 1×PCR buffer (Transgen Biotech) in a total volume of 20 μl. After that, an annealing procedure was performed: first, denaturation was carried out at 95°C for 5 minutes, then cooling from 95 to 20°C at a cooling rate of 1°C per minute, using a thermal cycler.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To a 20 μl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (final concentration of both components was 250 nM), target DNA (with a final concentration of 50 nM), fluorescent probe and quench probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. After that, it was inactivated by heating at 98°C for 5 minutes.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 µl of the inactivated reaction solution was added to a 96-well plate, and then detected using a microplate reader (535 nm excitation light, 556 nm emission light).

First of all, as shown in FIG. 7, for both the DNMT1-3 target sequence and the single-stranded DNA target, when the single-guide RNA guide sequence was 20 nt, the effect of base mutations at positions 1-12 on the fluorescent signal produced by trans- cleavage is was insignificant. When the guide length of the single guide RNA was 14 or 15 nt, the fluorescence values, including the control, were very low. When the length of the guide sequence of a single guide RNA was 16 nt, after the mutation of positions 10–12, the fluorescence value decreased significantly.

Upon further investigation, it was found that, as shown in FIG. 8, mutation of the base of positions 10-16 had an obvious effect on the fluorescence value, especially considering that the fluorescence value was almost undetectable after the mutation of positions 10-14. Then, different positions of the single-stranded target DNA were mutated into different types of bases, and this did not significantly affect the fluorescence value. However, only a mutation in the G position 9 significantly changed the fluorescence value (Fig. 9).

Then the present inventors also tested three other target sequences. Although different sequences had different fluorescence changes as a result of mutation of a specific site, the position resulting in a significant change could still be found for each sequence among positions 8-16, for example, positions 8, 10, 11 and 12 in rs5082, positions 11 in rs1467558, positions 8–15 in rs2952768 (Fig. 10).

In the case of the double-stranded DNA target in FIG. 11 shows that the fluorescent signal produced by the trans cleavage due to single base mutation exposure differs from the fluorescent signal of single stranded DNA. Using the target sequence DNMT1-3 as an example, a single guide RNA with a guide sequence length of 18–20 was susceptible to base mutations at positions 10–16. In addition, in this example, several types of single guide RNA are most sensitive to positions 10 and 16.

A one-step LAMP reaction and Cas12b protein detection were performed at 50°C, 55°C, 60°C and 65°C (FIG. 12).

Example 4

coli detection E. coli and other microorganisms in natural waters

The E. coli gyrB gene was chosen as a detection target for indirect detection of E. coli and other microorganisms in a water body and determination of the detection limit.

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as template and T7-crRNA-F was used as forward primer and ZL-gyrB-crRNA2-R was used as reverse primer. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 µl DpnI (10 U/µl) per 50 µl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The template DNA was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. LAMP amplification reaction

After culturing E. coli MG1655 to OD ₆₀₀ 1.0, a portion of the bacterial solution was subjected to a temperature of 100° C. for 15 minutes. After that, the LAMP amplification reaction was carried out. A bacterial solution of various concentrations was added to the matrix, while the diluted solutions had an optical density OD ₆₀₀ from 0.5×10 ^-3 , 5×10 ^-4 to 5×10 ^-9 , respectively.

After the coating test, OD ₆₀₀ 5×10 ^-3 contained about 7000 bacteria. The total volume of each reaction system was 25 μl. 1.6 µM LAMP-gyrB-FIP and LAMP-gyrB-BIP, 0.2 µM LAMP-gyrB-F3 and LAMP-gyrB-B3, and 0.4 µM LAMP-gyrB-LoopF and LAMP-gyrB-LoopB were used in as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To the 20 µl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (the final concentration of both components was 250 nM), 1 µl of LAMP product, fluorescent probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. After that, it was inactivated by heating at 98°C for 5 minutes.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

As shown in FIG. 13, the detection sensitivity of the Cas12b protein in combination with LAMP amplification (i.e., HOLMES v2.0 method) can reach the level of detection of very few E. coli (after coating test, OD ₆₀₀ = 5×10 ^-6 contained about 7 E. coli ).

Example 5 Saliva Sex Test

In this example, a specific gene locus of the sex-determining region of the Y chromosome was selected as a test target, and sex was determined using a saliva source sample by the HOLMES v2.0 method.

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as template and T7-crRNA-F was used as forward primer and ZL-sry-crRNA3-R and ZLsgRNA-DNMT1-3-R were used as reverse primers. The two DNA templates required for in vitro transcription were amplified by PCR, respectively. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The template DNA was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. LAMP amplification reaction

Samples of male and female saliva were used as templates and a LAMP amplification reaction was carried out. The total volume of each reaction system was 25 μl. 1.6 µM LAMP-sry-FIP and LAMP-sry-BIP, 0.2 µM LAMP-sry-F3 and LAMP-sry-B3, and 0.4 µM LAMP-sry-LoopF and LAMP-sry-LoopB were used in as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To the 20 µl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (the final concentration of both components was 250 nM), 1 µl of LAMP product, fluorescent probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. After that, it was inactivated by heating at 98°C for 5 minutes.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

As shown in FIG. 14, Cas12b protein combined with LAMP amplification (i.e. HOLMES v2.0 method) can effectively determine the sex of a person using saliva samples.

Example 6

Human SNP detection

In this example, the human rs5082 locus was detected. Target DNA was prepared by asymmetric PCR or isothermal LAMP amplification to determine the SNP type of samples obtained from saliva.

1. Preparation of guide RNA (single guide RNA)

The pUC18-sgRNA-DNMT1-3 plasmid was used as a template, T7-crRNA-F was used as a forward primer, and oligonucleotides containing guide sequences of various lengths complementary to the rs5082 site were used as reverse primers. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and the truncated single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C. In the case of target DNA generated by asymmetric PCR, a truncated single guide RNA with a guide sequence of 16 nt was used in the Cas12b protein detection reaction. In the case of a target DNA generated by LAMP amplification, a truncated single guide RNA with an 18 nt guide sequence was used in the Cas12b protein detection reaction.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. Amplification reaction (asymmetric PCR or LAMP amplification)

Asymmetric PCR amplification reaction: The human HEK293T cell genome was used as a template, and primers ASP primer, ASP-rs5082-F, ASP-rs5082-R were used for asymmetric PCR amplification (and traditional PCR does not use ASP primer), and the KOD FX enzyme was used for the amplification reaction.

LAMP amplification reaction: The human HEK293T cell genome was used as a template for the LAMP amplification reaction. The total volume of each reaction system was 25 μl. 1.6 µM LAMP-rs5082-FIP-10PAM (LAMP-rs5082-FIP as control) and LAMP-rs5082-BIP, 0.2 µM LAMP-rs5082-F3 and LAMP-rs5082-B3, and 0.4 µM LAMP -rs5082-LoopF and LAMP-rs5082-LoopB were used as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction. Since a PAM sequence was added to the LAMP-rs5082-FIP-10PAM primer sequence, the amplified DNA has a PAM sequence.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b reaction: The mixture of single guide RNA and Cas12b incubated in step (2) was added to the 20 µl reaction system (the final concentration of both components was 500 nM, and the concentration of Cas12b in the LAMP amplification test was 250 nM), 1 µl target DNA or 1 µl LAMP product, fluorescent probe (HEX-N12-BHQ1 final concentration 500 nM) and 2 µl buffer 3.1 10×NEB and 0.5 µl RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 60 minutes. Thereafter, it was inactivated by heating at 98°C for 5 minutes in a PCR instrument.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

From FIG. 15 shows that since there is no PAM sequence near the rs5082 site, asymmetric PCR that produces single-stranded DNA can recognize SNP sites more clearly.

When combined with LAMP amplification, since there was no PAM sequence near the rs5082 site, the detection site must be introduced with a PAM sequence for better recognition of the SNP sites (FIG. 16).

Example 7

RNA virus detection

In this example, JEV (Japanese Encephalitis Virus) RNA was selected and the HOLMES v2.0 method was used to determine if a sample was infected with a virus, and sites E453 and NS170 were selected for testing.

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as a template, T7-crRNA-F was used as a forward primer, and oligonucleotides containing guide sequences complementary to the respective targets were used as reverse primers. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. LAMP amplification reaction

Cell fluid infected with JEV virus was used as a template, and a LAMP amplification reaction was carried out. The total volume of each reaction system was 25 μl. 1.6 µM LAMP-E453-FIP and LAMP-E453-BIP, 0.2 µM LAMP-E453-F3 and LAMP-E453-B3, and 0.4 µM LAMP-E453-LoopF and LAMP-E453-LoopB (or 1, 6 μM LAMP-NS170-FIP and LAMP-NS170-BIP, 0.2 μM LAMP-NS170-F3 and LAMP-NS170-B3, and 0.4 μM LAMP-NS170-LoopF and LAMP-NS170-LoopB) were used as primers. For LAMP amplification, the WarmStart® LAMP Kit (NEB) or Bst 3.0 DNA polymerase (NEB) was used. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To the 20 µl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (the final concentration of both components was 250 nM), 1 µl of LAMP product, fluorescent probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. Thereafter, it was inactivated by heating at 98°C for 5 minutes in a PCR instrument.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light).

As shown in FIG. 17, the Cas12b protein combined with LAMP amplification (i.e. HOLMES v2.0 method) can detect the presence of the JEV virus with a high degree of sensitivity.

Example 8

Relative quantification of trace DNA template (real-time HOLMES method)

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as a template, T7-crRNA-F was used as a forward primer, and oligonucleotides containing guide sequences complementary to the targets were used as reverse primers. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

DNase I was then added to the transcription system (2 µl DNase I (5 U/µl) per 50 µl transcription system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. One-step LAMP-amplification-fluorescence detection reaction (using a real-time PCR instrument)

Plasmids DNMT1-3 of various concentrations were used as templates for the LAMP amplification reaction. The total volume of each reaction system was 20 μl. 1.6 µM LAMP-DNM-FIP and LAMP-DNM-BIP, 0.2 µM LAMP-DNM-F3 and LAMP-DNM-B3, and 0.4 µM LAMP-DNM-LoopF and LAMP-DNM-LoopB were used in as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. A mixture of single guide RNA and Cas12b (final concentration of both components was 500 nM), a fluorescent probe (FAM-N12-Eclipse with a final concentration of 500 nM) and 0.5 μl of RNase inhibitor (40 U/μl) were added. After mixing in a fluorometer, fluorescence detection was performed and the reaction proceeded at 55° C. for 120 minutes.

3. Analysis of results

In FIG. 18 shows real-time target DNA fluorescence measurements at various concentrations.

An approximate linear trend was obtained, using the time when the fluorescence value reaches 600,000 as the y-axis, and using the absolute value of the lg concentration as the x-axis.

The above results show that the method of the present invention can accurately detect 10 ^-1 -10 ^-6 nM DNA within 45 minutes.

Example 9

Absolute quantitation of trace DNA template (HOLMES digital method)

In this example, the HOLMES method was combined with digital PCR on chips for absolute quantitation at the same reaction temperature.

1. Preparation of guide RNA (single guide RNA)

The pUC18-sgRNA-DNMT1-3 plasmid was used as a template, T7-crRNA-F was used as a forward primer, and oligonucleotides containing guide sequences of various lengths complementary to the rs5082 site were used as reverse primers. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to this PCR product (1 µl DpnI (10 U/µl) per 50 µl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. One-Step LAMP Amplification-Fluorescence Detection Reaction (Using JN Medsys Digital Drop PCR (ddPCR) Instrument)

The 293T human cell genome at various concentrations was used as a matrix for absolute quantitation. The total volume of each reaction system was 15 μl. 1.6 µM LAMP-rs5082-FIP-10PAM and LAMP-rs5082-BIP, 0.2 µM LAMP-rs5082-F3 and LAMP-rs5082-B3, and 0.4 µM LAMP-rs5082-LoopF and LAMP-rs5082-LoopB were used as primers. The WarmStart® LAMP Kit (NEB) was used for the LAMP reaction. A mixture of single guide RNA and Cas12b (final concentration of both components was 500 nM), fluorescent probe (HEX-N12-BHQ1 and FAM-N12-Eclipse, both with a final concentration of 500 nM), 0.375 μl of RNase inhibitor (40 U/μl ) and 0.75 µl of JN solution.

After mixing with a glass slide, the sample was taken to the chip (in a PCR tube) to split the system. The chip was then transferred to the Sealing Enhancer for processing.

Then 235 µl of blocking solution was added and the reaction proceeded in a metal bath at 55° C. for 120 minutes. At 0, 60, 90, and 120 minutes, the chip was placed in the View Jig for fluorescence detection.

3. Analysis of results

As shown in FIG. 20, after 120 minutes of reaction, 51.75 copies were found by counting in template 1 and 6.3 copies were found in template 2 at 10-fold dilution. No copies were found in the no-matrix control.

The results show that the digital HOLMES method of the present invention allows not only to quantify but also accurately detect a single copy of a template in a micro-detection system with exceptional sensitivity, while saving significant time.

Example 10

Detection of methylation sites

In this example, a 250 bp promoter region of the α2(I) collagen gene ( COL1A2 ) containing 13 CpG sites (M1-M13) was selected as the sequence for target gene detection (accession number NCBI: AF004877.1). The α2(I) collagen gene ( COL1A2 ) codes for collagen and is related to oncogenesis.

In COL1A2(BSP)-C, all CpG sites were modified by methylation. After bisulfite conversion (conversion of unmethylated C to uracil) and PCR amplification, the methylated base C in CpG still remained C.

In COL1A2(BSP)-T, none of the CpG sites were modified by methylation. After bisulfite conversion and PCR amplification, base C at the CpG site is converted to T.

The principle of the methylation site detection method of the present invention is shown in FIG. 21.

Experimental way

1. The genomes of four cancer cell lines (293T, SW480, NCI-N87 and MCF-7) were extracted.

2. The genome was subjected to bisulfite conversion. The promoter region of the COL1A2 gene containing the M3 site was selected and the corresponding BSP primers were designed (primer COL1A2(BSP)-F/R). The bisulfite-converted cell genome was used as a template for PCR amplification, and the gel was reconstituted and purified to obtain a product that was used as a DNA target for subsequent detection.

3. Preparation of the target nucleic acid for the calibration curve

(1) Primers (primer COL1A2-F/R) were used to amplify the COL1A2 gene fragment from SW480-gDNA, and it was assembled into pClone007S T vector (TSINGKE, TSV-007S). The vector was then transformed into DH10b. After culturing in LB medium with appropriate antibiotics at 37°C, the plasmid was recovered and sequenced, and finally plasmid pClone007-COL1A2 was obtained.

(2) Plasmid pClone007-COL1A2 was treated with CpG methyltransferase (M.SssI) (M0226V, NEB) so that cytosine residues in all 5'-CG-3' sequences were methylated. Thereafter, plasmids pClone007-COL1A2 and pClone007-COL1A2 treated with CpG methyltransferase were subjected to a bisulfite conversion to convert unmethylated cytosine to uracil. A pair of BSP primers (primer COL1A2(BSP)-F/R) for PCR amplification was designed and the amplified fragments were repaired and gel-purified and assembled into pClone007S T vector to construct plasmids pClone007-COL1A2(BSP)-C and pClone007- COL1A2(BSP)-T, thus obtaining COL1A2(BSP)-C (Target C) and COL1A2(BSP)-T (Target T) fragments.

4. Methylation detection

Four gDNA cancer cell lines (293T, SW480, NCI-N87 and MCF-7) were treated with the EZ DNA methylation-direct ^TM kit (D5021, Zymo Research) for bisulfite conversion. After that, a pair of BSP primers (primer COL1A2(BSP)-F/R) was used to amplify fragments of the COL1A2(BSP) gene (250bp) of four cancer cell lines, and gel isolation and purification were performed.

Clone bisulfite sequencing method

The above four COL1A2 (BSP) gene fragments were fused to the pClone007S T vector and then transformed into DH10b. Over 10 positive monoclonal clones were collected per plate and sent for sequencing analysis. A methylation rate calculation tool, QUantification tool for Methylation Analysis (QUMA) was used to calculate the degree of methylation of 13 CpG sites in the COL1A2 gene.

Direct bisulfite sequencing

The above four fragments of the COL1A2(BSP) gene were analyzed by the Sanger sequencing method, and the methylation rate was obtained by calculating the C/(C+T) peak ratio of 13 CpG sites in the COL1A2 gene.

High throughput next generation bisulfite sequencing (NGS) method

The above four COL1A2 (BSP) gene fragments were subjected to high throughput NGS bisulfite sequencing, and high throughput NGS bisulfite sequencing (conducted by Zhongke Purui Biotechnology Co., Ltd.) and the methylation degree of 13 CpG sites in the COL1A2 gene were analyzed.

Methylation detection with the HOLMESv2 system

(1) Extraction of CRISPR-Cas12b protein

A codon-optimized full-length AacCas12b gene was synthesized by Shanghai Tolo Biotechnology Company Limited and cloned into the pET28a vector. The HIS-tagged N-terminal recombinant Cas12b protein was expressed and produced in E. coli BL21 (DE3). When the OD ₆₀₀ of the bacterial solution reached 0.6, 0.25 mM isopropyl thiogalactoside (IPTG) was added to induce expression, and the bacterial solution was cultured at 16°C for 14–18 h. After that, the solution was centrifuged to collect the bacteria. The bacterial solution was suspended in buffer A (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 20 mM imidazole and 1/500 toluenesulfonyl fluoride (v/v)) and lysed with a high pressure homogenizer (Avestin), and centrifuged at 15,000 rpm for 30 minutes. The Ni-column (GE Healthcare) was pre-soaked in buffer A and the supernatant was transferred to the Ni-column. After that, buffer B (50 mM Tris-HCl (pH 7.6), 200 mM NaCl and 30 mM imidazole) was used to elute the Ni column, and buffer B (in which the concentration of imidazole was adjusted to 300 mM) was used to to elute the recombinant protein. The eluted fractions containing the AacCas12b protein were collected by ultrafiltration and concentrated to approximately 5 ml. It was then loaded onto a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) maintained in buffer C (40 mM Tris-HCl (pH 7.6), 200 mM NaCl, 2 mM DTT, and 5% glycerol (v/v). vol))), and Eluted fractions containing AacCas12b protein were pooled, concentrated to 4 mg/ml with 50% glycerol added, and stored at -20°C.

(2) Preparation of single guide RNA

Plasmid pUC18-sgRNA-DNMT1-3 was used as a template and T7-sgRNA-F was used as a forward primer, and target-specific reverse primers were designed. The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to this PCR product (1 μl DpnI per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C. Finally, DNase I was added to the transcription system (2 μl DNase I per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The template DNA was removed and an RNA purification and isolation kit (Zymo Research) was used to purify the RNA. The resulting single guide RNA was named sgRNA-DNMT1-3. The amount of product was determined using NanoDrop 2000C and the product was placed in a refrigerator for storage at -80°C for later use.

(3) Single guide RNA screening

In the case of COL1A2, a plurality of single guide RNAs were constructed, in which guide sequences of varying lengths coincided with regions above and below each CpG site, and CpG sites coincided with various positions of the gene. A single guide RNA (with a final concentration of 10 μM) was annealed in a PCR instrument. The annealing procedure was as follows: 75°C for 5 minutes; then cooling at a rate of 1°C in 1 minute to 20°C. After annealing, the single guide RNA and the AacCas12b protein (with a final concentration of 5 µM) were mixed in equal volumes and incubated at 30°C for 15–20 minutes. After that, the 20 µl Cas12b reaction system was ready (work on ice):

Cas12b 250 nM Single guide RNA 500 nM Target DNA (COL1A2(BSP)-C/T) 100 nM HEX-N12-BHQ1 (fluorescent labeling) 500 nM 10 × NEB buffer 3.1 2 µl RRI (RNase Inhibitor) 0.5 µl

RNase-free water was added to a total volume of 20 µl.

The reaction proceeded in a water bath at 48° C. for 1 hour and 20 µl of the system was transferred to a 96-well plate. On the microplate reader, the excitation light wavelength was set to 535 nm and the emission light wavelength was set to 556 nm to measure the fluorescence value. The most appropriate single guide RNA used in the reaction group was selected, in which the target DNA (COL1A2(BSP)-C/T) had the greatest difference in fluorescence value.

(4) Methylation detection based on the HOLMESv2 system

Fragments COL1A2(BSP)-C and COL1A2(BSP)-T were thoroughly mixed according to the ratio of COL1A2(BSP)-C as 0%, 10%, 30% and 50%, respectively, as a target DNA methylation calibration curve. Bisulfite-treated probe COL1A2 (BSP) fragments from four cancer cell lines (293T, SW480, NCI-N87, and MCF-7) were mixed with an equimolar concentration of COL1A2 (BSP)-T, respectively, and the mixtures were then used as DNA targets for determining the methylation ratio of real samples. Then, the methylation calibration curve of the M3 site of the COL1A2 promoter region and the ratio of M3 site methylation in four cancer cell lines (293T, SW480, NCI-N87, MCF-7) were determined, respectively, using sgRNA-COL1A2m3-C12-17 in the HOLMESv2 system.

A single guide RNA (with a final concentration of 10 μM) was annealed in a PCR instrument. The annealing procedure was as follows: 75°C for 5 minutes; then cooling at a rate of 1 °C in 1 minute to 20 °C. After annealing, single guide RNA and AacCas12b protein (final concentration 5 µM) were mixed in equal volumes and incubated at 30°C for 15–20 minutes. After that, the 20 µl Cas12b reaction system was ready (work on ice):

Cas12b 250 nM Single guide RNA 500 nM DNA target (M3) 5 nM FAM-N12-Eclipse (fluorescent label) 500 nM DNA from salmon milt fragmented into result of hydrodynamic shear 500 ng 10 × NEB buffer 3.1 2 µl RRI (RNase Inhibitor) 0.25 µl

RNase-free water was added to a total volume of 20 µl.

20 µl of the system was transferred to a 96-well plate, which was then placed in a real-time PCR instrument. The program was set to run the reaction at 48 C for 1 hour, and the fluorescence value was measured every 2 minutes.

The calibration curve for methylation at the M3 site took the fluorescence value of 30 minutes and 10 minutes as the Y ordinate, and took the COL1A2(BSP)-C ratio of 0%, 10%, 30% and 50% as the X abscissa. The equation model was as follows: Y =aX+b. The concentration of COL1A2 (BSP) in four lines of cancer cells was 1/2 of the target DNA on the calibration curve. Thus, the model for calculating the methylation ratio took the form: X _unknown =2(Y _unknown -b)/a.

Experimental results

1. Diagram of detection mode

After the genes containing methylated CpG sites were subjected to bisulfite conversion, all unmethylated CG sites in the genes were converted to UG, and all methylated CG sites remained unchanged. The transformed gene was used as a DNA template for the formation of a triple complex with the AacCas12b protein and a single guide RNA. In a single-stranded DNA probe system with a fluorescent group and a quench group (i.e., the HOLMESv2 system), the reaction was constant at 48°C for a certain period, and the fluorescence value was determined. If the detection site is methylated, the DNA probe emits light and the fluorescence value is high. If the detection site is not methylated, the DNA probe does not emit light and the fluorescence value is low or zero.

2. Single guide RNA screening

As shown in FIG. 22, by comparing the fluorescence values of each single guide RNA and two targets (target-C and target-T represent COL1A2 fragments after bisulfite treatment with methylation at all CpG sites of 100% and 0%, respectively) in the HOLMESv2-Cas12b reaction system, one can choose the most a suitable single guide RNA. At the same time, the fluorescence values of 9 sgRNAs, m3-C12-17, m4-C1-18, m4-C1-19, m4-C1-20, m5-C14-18, m6m7m8-C7-18, m6m7m8-19, m6m7m8- 20 and m9m10-C1-18 had obvious differences in two targets (target-C, target-T), all of which can be used to detect methylation. In this study, a single point single guide RNA (m3-C12-17) was selected for subsequent methylation detection. A single RNA is shown in Fig. 23.

3. Quantification of methylation at the CpG site M3 ( COL1A2 )

Fragments of COL1A2 (BSP)-C and COL1A2 (BSP)-T were mixed according to the content ratio of COL1A2 (BSP)-C 0%, 10%, 30%, 50% and sgRNA-COL1A2m3 -C12-17 used to determine the fluorescence value reactions after 30 minutes in the HOLMESv system. After data analysis, a fluorescence value of 0% was used as the zero point for normalization, and a quantitative calibration curve of the CpG site M3 (COL1A2) was constructed. A calibration curve was constructed accordingly for each test. The authors of the present invention performed each test three times with three repetitions. The calibration curve equations were as follows: y = 758939x + 9320.9 (R ² = 0.9975); y = 671675x + 12415 (R ² = 0.9943); and y = 645621x + 8601.7 (R ² = 0.997), respectively. The linear correlation of the three calibration curves was very good, with R ² reaching a value of 0.99 or more. Thus, the fluorescence value of a given gene locus in real samples can be determined using the HOLMESv2 system to calculate the corresponding degree of methylation (Fig. 25).

COL1A2 demonstrated different levels of methylation in different forms of cancer. To test the applicability of the methylation detection method using the HOLMESv2-Cas12b system, sgRNA-COL1A2m3-C12-17 was used to determine the fluorescence values of the CpG site M3 (COL1A2) in four types of cancer cells (293T, SW480, NCI-N87 and MCF-7) through 30 minutes of reaction in the HOLMESv2 system (Fig. 24). At the same time, the concentration of the target gene found in cancer cells was 1/2 of the concentration of the calibration curve. Their degrees of methylation were calculated from the above equations of the calibration curve, respectively: 63.3%, 81.3%, 54.8% and 77.1%.

In addition, the widely used methylation detection method was simultaneously used to determine the methylation levels of the COL1A2 gene in the above four cancer cells, and compared with the method of the present invention to test the efficiency and applicability of the methylation detection method using the HOLMESv2-Cas12b system (Fig. 26) .

The high throughput new generation bisulfite sequencing (NGS) method can be used to quantify methylation. The methylation rates of the CpG site M3 ( COL1A2 ) in four cancer cells (293T, SW480, NCI-N87, and MCF-7) were: 65.26%, 81.06%, 48.27%, and 67.3%, corresponding to detection results obtained using the HOLMESv2-Cas12b system.

At present, the direct bisulfite sequencing method and the clone bisulfite sequencing method can only be used for semi-quantitative methylation. The results of detection of methylation at the CpG site M3 ( COL1A2 ) in the above four cancer cells (293T, SW480, NCI-N87 and MCF-7) by these two methods were 63.2%, 63.6%, 30.4%, 20% and 92.3% (12/13), 100% (15/15), 78.6% (11/14), 71.4% (10/14). Compared to the results obtained by the NGS high-throughput bisulfite sequencing method, the difference was large, with the former being low and the latter being high.

The above results show that the quantitative results obtained by the detection method using the HOLMESv2-Cas12b system of the present invention are very accurate and very convenient and time-consuming to obtain.

Example 11

Detection of alternatively spliced RNA and circular RNA

Alternative pre-mRNA splicing is a key link in the post-transcriptional regulation of high eukaryotes and plays an important role in human diseases, such as tumor progression. Some pre-mRNAs can be processed to form circular RNAs.

In this example, the HOLMESv2 system was used to detect mRNA splicing specifically targeting the splice site to detect, in combination with RT-LAMP amplification, the spliced mature mRNA of the GAPDH gene (FIG. 27). In addition, a similar strategy was adopted in the construction of crRNA, and the Cdr1as cRNA in HEK293T was successfully detected in this example (FIG. 27).

1. Preparation of guide RNA (single guide RNA)

Plasmid pUC18-sgRNA-DNMT1-3 was used as a template, T7-sgRNA-F was used as a forward primer, and oligonucleotides containing guide sequences complementary to the respective targets were used as reverse primers (see Table 2). The template DNA required for in vitro transcription was amplified by PCR. DpnI was then added to the PCR product (1 μl DpnI (10 U/μl) per 50 μl PCR system), incubated in a water bath at 37°C for 30 minutes. The plasmid DNA template was digested and a (Promega) column kit was used to recover the PCR product from the gel. The reconstituted PCR product was used as a template and a single guide RNA was synthesized using the T7 High Yield Transcription Kit (Thermo). The reaction proceeded at night (12–16 h) at a temperature of 37°C.

Then DNase I was added to the transcription system (2 μl DNase I (5 U/μl) per 50 μl transcription system), incubated in a water bath at 37°C for 30 minutes. The template DNA was removed and an RNA purification and isolation kit was used to purify the RNA. The NanoDrop 2000C was then used for quantitation and the product was placed in a refrigerator for storage at -80°C for later use.

2. LAMP amplification reaction

The 293T cell genome was used to carry out the LAMP amplification reaction. The total volume of each reaction system was 25 μl. 1.6 μM LAMP-FIP and LAMP-BIP, 0.2 μM LAMP-F3 and LAMP-B3, and 0.4 μM LAMP-LoopF and LAMP-LoopB were used as primers. For LAMP amplification, the WarmStart® LAMP Kit (NEB) or Bst 3.0 DNA polymerase (NEB) was used. The LAMP reaction proceeded at 65°C for 30 minutes. After completion of the LAMP reaction, the product was inactivated at 85°C for 10 minutes and then directly used in the Cas12b reaction.

3. Cas12b reaction

(1) Single guide RNA annealing: The single guide RNA was diluted to an appropriate concentration (5 μM) and annealed in a PCR instrument. Annealing procedure: denaturation at 75°C for 5 minutes, then cooling from 75 to 20°C at a cooling rate of 1°C per minute.

(2) Incubation of single guide RNA with Cas12b: The annealed single guide RNA was incubated with Cas12b at an equimolar concentration and placed at 30°C for 20–30 minutes.

(3) Cas12b Reaction: To the 20 µl reaction system, the mixture of single guide RNA and Cas12b incubated in step (2) (the final concentration of both components was 250 nM), 1 µl of LAMP product, fluorescent probe (HEX-N12-BHQ1 with a final concentration of 500 nM) and 2 µl of buffer 3.1 10×NEB and 0.5 µl of RNase inhibitor (40 U/µl). After mixing, the reaction proceeded at 48°C for 30 minutes. Thereafter, it was inactivated by heating at 98°C for 5 minutes in a PCR instrument.

4. Detection of the bypass cleavage activity of single-stranded DNA of the Cas12b protein using a fluorescent microplate reader.

20 μl of the inactivated reaction solution was added to a 96-well plate and detected using a microplate reader (535 nm excitation light, 556 nm emission light). In FIG. 27 shows that after LAMP amplification, Cas12b (ie HOLMES v2.0 method) can detect and determine whether the expected RNA splicing has occurred or whether it is a circular RNA.

Table 1. Oligonucleotides as substrates

Name Sequence (5'-3') Id. Sequence No.: target-T1-F tttctgtttgttatcgcaactttctactgaattcaagctttactctagaaagaggagaaaggatcc 1 target-T1-R ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagttgcgataacaaacagaaa 2 target-T1-FAM-5'-R fam-ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagttgcgataacaaacagaaa 3 target-T1-R-FAM ggatcctttctcctctttctagagtaaagcttgaattcagtagaaagttgcgataacaaacagaaa-fam 4 DNMT1-3(TTC PAM)-F aatgttccctgatggtccatgtctgttactcgcctgtcaagtggcgtgac 5 DNMT1-3(TTC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggaacatt 6 DNMT1-3(AAC PAM)-F aatgaaccctgatggtccatgtctgttactcgcctgtcaagtggcgtgac 7 DNMT1-3(AAC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggttcatt 8 DNMT1-3(ATC PAM)-F aatgatccctgatggtccatgtctgttactcgcctgtcaagtggcgtgac 9 DNMT1-3(ATC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggatcatt 10 DNMT1-3(TAC PAM)-F aatgtaccctgatggtccatgtctgttactcgcctgtcaagtggcgtgac eleven DNMT1-3(TAC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggtacatt 12 DNMT1-3(GGC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggcccatt 13 DNMT1-3(CCC PAM)-R gtcacgccacttgacaggcgagtaacagacatggaccatcagggggcatt 14 DNMT1-3-C1G-F aatgttcgctgatggtccatgtctgttactcgcctgtcaagtggcgtgac 15 DNMT1-3-C1G-R gtcacgccacttgacaggcgagtaacagacatggaccatcagcgaacatt 16 DNMT1-3-C2G-F aatgttccgtgatggtccatgtctgttactcgcctgtcaagtggcgtgac 17 DNMT1-3-C2G-R gtcacgccacttgacaggcgagtaacagacatggaccatcacggaacatt 18 DNMT1-3-T3A-F aatgttcccagatggtccatgtctgttactcgcctgtcaagtggcgtgac 19 DNMT1-3-T3A-R gtcacgccacttgacaggcgagtaacagacatggaccatctgggaacatt 20 DNMT1-3-G4C-F aatgttccctcatggtccatgtctgttactcgcctgtcaagtggcgtgac 21 DNMT1-3-G4C-R gtcacgccacttgacaggcgagtaacagacatggaccatgagggaacatt 22 DNMT1-3-A5T-F aatgttccctgttggtccatgtctgttactcgcctgtcaagtggcgtgac 23 DNMT1-3-A5T-R gtcacgccacttgacaggcgagtaacagacatggaccaacagggaacatt 24 DNMT1-3-T6A-F aatgttccctgaaggtccatgtctgttactcgcctgtcaagtggcgtgac 25 DNMT1-3-T6A-R gtcacgccacttgacaggcgagtaacagacatggaccttcagggaacatt 26 DNMT1-3-G7C-F aatgttccctgatcgtccatgtctgttactcgcctgtcaagtggcgtgac 27 DNMT1-3-G7C-R gtcacgccacttgacaggcgagtaacagacatggacgatcagggaacatt 28 DNMT1-3-G8C-F aatgttccctgatgctccatgtctgttactcgcctgtcaagtggcgtgac 29 DNMT1-3-G8C-R gtcacgccacttgacaggcgagtaacagacatggagcatcagggaacatt thirty DNMT1-3-T9A-F aatgttccctgatggaccatgtctgttactcgcctgtcaagtggcgtgac 31 DNMT1-3-T9A-R gtcacgccacttgacaggcgagtaacagacatggtccatcagggaacatt 32 DNMT1-3-C10G-F aatgttccctgatggtgcatgtctgttactcgcctgtcaagtggcgtgac 33 DNMT1-3-C10G-R gtcacgccacttgacaggcgagtaacagacatgcaccatcagggaacatt 34 DNMT1-3-C11G-F aatgttccctgatggtcgatgtctgttactcgcctgtcaagtggcgtgac 35 DNMT1-3-C11G-R gtcacgccacttgacaggcgagtaacagacatcgaccatcagggaacatt 36 DNMT1-3-A12T-F aatgttccctgatggtccttgtctgttactcgcctgtcaagtggcgtgac 37 DNMT1-3-A12T-R gtcacgccacttgacaggcgagtaacagacaaggaccatcagggaacatt 38 DNMT1-3-T13A-F aatgttccctgatggtccaagtctgttactcgcctgtcaagtggcgtgac 39 DNMT1-3-T13A-R gtcacgccacttgacaggcgagtaacagacttggaccatcagggaacatt 40 DNMT1-3-G14C-F aatgttccctgatggtccatctctgttactcgcctgtcaagtggcgtgac 41 DNMT1-3-G14C-R gtcacgccacttgacaggcgagtaacagagatggaccatcagggaacatt 42 DNMT1-3-T15A-F aatgttccctgatggtccatgactgttactcgcctgtcaagtggcgtgac 43 DNMT1-3-T15A-R gtcacgccacttgacaggcgagtaacagtcatggaccatcagggaacatt 44 DNMT1-3-C16G-F aatgttccctgatggtccatgtgtgttactcgcctgtcaagtggcgtgac 45 DNMT1-3-C16G-R gtcacgccacttgacaggcgagtaacacacatggaccatcagggaacatt 46 DNMT1-3-T17A-F aatgttccctgatggtccatgtcagttactcgcctgtcaagtggcgtgac 47 DNMT1-3-T17A-R gtcacgccacttgacaggcgagtaactgacatggaccatcagggaacatt 48 target-01-rs-R agacttagatctgagccctccctcttcccagcacaggcaggggtagaagc 49 target-01-rs-12T-R agacttagatctgagccctccctcttcccagaacaggcaggggtagaagc 50 target-02-rs-R tgtgtttggatttgcagtaggctgaagcgttatactatgactggagtcca 51 target-02-rs-12C-R tgtgtttggatttgcagtaggctgaagcgttgtactatgactggagtcca 52 target-03-rs-R aaaatagtgctttttacttttatctgaatgattgaaatgtccttttccca 53 target-03-rs-12C-R aaaatagtgctttttactttttctgaatgactgaaatgtccttttccca 54 DNMT1-3-C1A-R gtcacgccacttgacaggcgagtaacagacatggaccatcagtgaacatt 55 DNMT1-3-C1T-R gtcacgccacttgacaggcgagtaacagacatggaccatcagagaacatt 56 DNMT1-3-C2T-R gtcacgccacttgacaggcgagtaacagacatggaccatcaaggaacatt 57 DNMT1-3-C2A-R gtcacgccacttgacaggcgagtaacagacatggaccatcatggaacatt 58 DNMT1-3-T3G-R gtcacgccacttgacaggcgagtaacagacatggaccatccgggaacatt 59 DNMT1-3-T3C-R gtcacgccacttgacaggcgagtaacagacatggaccatcggggaacatt 60 DNMT1-3-G4T-R gtcacgccacttgacaggcgagtaacagacatggaccataagggaacatt 61 DNMT1-3-G4A-R gtcacgccacttgacaggcgagtaacagacatggaccattagggaacatt 62 DNMT1-3-A5G-R gtcacgccacttgacaggcgagtaacagacatggaccaccagggaacatt 63 DNMT1-3-A5C-R gtcacgccacttgacaggcgagtaacagacatggaccagcagggaacatt 64 DNMT1-3-T6G-R gtcacgccacttgacaggcgagtaacagacatggaccctcagggaacatt 65 DNMT1-3-T6C-R gtcacgccacttgacaggcgagtaacagacatggaccgtcagggaacatt 66 DNMT1-3-G7T-R gtcacgccacttgacaggcgagtaacagacatggacaatcagggaacatt 67 DNMT1-3-G7A-R gtcacgccacttgacaggcgagtaacagacatggactatcagggaacatt 68 DNMT1-3-G8T-R gtcacgccacttgacaggcgagtaacagacatggaacatcagggaacatt 69 DNMT1-3-G8A-R gtcacgccacttgacaggcgagtaacagacatggatcatcagggaacatt 70 DNMT1-3-T9G-R gtcacgccacttgacaggcgagtaacagacatggcccatcagggaacatt 71 DNMT1-3-T9C-R gtcacgccacttgacaggcgagtaacagacatgggccatcagggaacatt 72 DNMT1-3-C10T-R gtcacgccacttgacaggcgagtaacagacatgaaccatcagggaacatt 73 DNMT1-3-C10A-R gtcacgccacttgacaggcgagtaacagacatgtaccatcagggaacatt 74 DNMT1-3-C11T-R gtcacgccacttgacaggcgagtaacagacatagaccatcagggaacatt 75 DNMT1-3-C11A-R gtcacgccacttgacaggcgagtaacagacattgaccatcagggaacatt 76 DNMT1-3-A12G-R gtcacgccacttgacaggcgagtaacagacacggaccatcagggaacatt 77 DNMT1-3-A12C-R gtcacgccacttgacaggcgagtaacagacagggaccatcagggaacatt 78 DNMT1-3-T13G-R gtcacgccacttgacaggcgagtaacagacctggaccatcagggaacatt 79 DNMT1-3-T13C-R gtcacgccacttgacaggcgagtaacagacgtggaccatcagggaacatt 80 DNMT1-3-G14T-R gtcacgccacttgacaggcgagtaacagaaatggaccatcagggaacatt 81 DNMT1-3-G14A-R gtcacgccacttgacaggcgagtaacagatatggaccatcagggaacatt 82 DNMT1-3-T15G-R gtcacgccacttgacaggcgagtaacagccatggaccatcagggaacatt 83 DNMT1-3-T15C-R gtcacgccacttgacaggcgagtaacaggcatggaccatcagggaacatt 84 DNMT1-3-C16T-R gtcacgccacttgacaggcgagtaacaaacatggaccatcagggaacatt 85 DNMT1-3-C16A-R gtcacgccacttgacaggcgagtaacatacatggaccatcagggaacatt 86 target-01rs-C8G-R agacttagatctgagccctccctcttcccagcacacgcaggggtagaagc 87 target-01rs-T9A-R agacttagatctgagccctccctcttcccagcactggcaggggtagaagc 88 target-01rs-G10C-R agacttagatctgagccctccctcttcccagcagaggcaggggtagaagc 89 target-01rs-T11A-R agacttagatctgagccctccctcttcccagctcaggcaggggtagaagc 90 target-01rs-G12C-R agacttagatctgagccctccctcttcccaggacaggcaggggtagaagc 91 target-01rs-C13G-R agacttagatctgagccctcccttcccaccacaggcaggggtagaagc 92 target-01rs-T14A-R agacttagatctgagccctccctcttccctgcacaggcaggggtagaagc 93 target-01rs-G15C-R agacttagatctgagccctccctcttccgagcacaggcaggggtagaagc 94 target-01rs-G16C-R agacttagatctgagccctccctcttcgcagcacaggcaggggtagaagc 95 target-02rs-A8T-R tgtgtttggatttgcagtaggctgaagcgttatacaatgactggagtcca 96 target-02rs-G9C-R tgtgtttggatttgcagtaggctgaagcgttatagtatgactggagtcca 97 target-02rs-T10A-R tgtgtttggatttgcagtaggctgaagcgtttattctatgactggagtcca 98 target-02rs-A11T-R tgtgtttggatttgcagtaggctgaagcgttaaactatgactggagtcca 99 target-02rs-T12A-R tgtgtttggatttgcagtaggctgaagcgttttactatgactggagtcca 100 target-02rs-A13T-R tgtgtttggatttgcagtaggctgaagcgtaatactatgactggagtcca 101 target-02rs-A14T-R tgtgtttggatttgcagtaggctgaagcgatatactatgactggagtcca 102 target-02rs-C15G-R tgtgtttggatttgcagtaggctgaagccttatactatgactggagtcca 103 target-02rs-G16C-R tgtgtttggatttgcagtaggctgaagggttatactatgactggagtcca 104 target-03rs-T8A-R aaaatagtgctttttacttttatctgaatgattgatatgtccttttccca 105 target-03rs-T9A-R aaaatagtgctttttactttttctgaatgattgtaatgtccttttccca 106 target-03rs-C10G-R aaaatagtgctttttacttttatctgaatgattcaaatgtccttttccca 107 target-03rs-A11T-R aaaatagtgctttttactttttctgaatgatagaaatgtccttttccca 108 target-03rs-A12T-R aaaatagtgctttttactttttctgaatgaatgaaatgtccttttccca 109 target-03rs-T13A-R aaaatagtgctttttactttttctgaatgtttgaaatgtccttttccca 110 target-03rs-C14G-R aaaatagtgctttttactttttctgaatcattgaaatgtccttttccca 111 target-03rs-A15T-R aaaatagtgctttttacttttatctgaaagattgaaatgtccttttccca 112 target-03rs-T16A-R aaaatagtgctttttacttttatctgattgattgaaatgtccttttccca 113 target-05rs-R ggatctcctggcggaggtggtggtagaaggtccaggagcaggggtagccg 114 target-05rs-G11A-R ggatctcctggcgggaggtggtggtagaaggtctaggagcaggggtagccg 115 target-41rs-R agcacatgtttgttgaatgagtcagtggatgacgtagatcagtatcttgt 116 target-41rs-G11A-R agcacatgtttgttgaatgagtcagtggatgatgtagatcagtatcttgt 117 target-01rs-10SNP-F gcttctttccctgcctgtgctgggaagagggaggggctcagatctaagtct 118 target-01rs-10SNP-R agacttagatctgagccctccctcttcccagcacaggcagggaaagaagc 119 target-01rs-10SNP-G10T-F gcttctttccctgcctgttctgggaagagggaggggctcagatctaagtct 120 target-01rs-10SNP-G10T-R agacttagatctgagccctccctcttcccagaacaggcagggaaagaagc 121 target-01rs-12SNP-F gcttttacccctgcctgtgctgggaagagggaggggctcagatctaagtct 122 target-01rs-12SNP-R agacttagatctgagccctccctcttcccagcacaggcaggggtaaaagc 123 target-01rs-12SNP-G12T-F gcttttacccctgcctgttctgggaagagggaggggctcagatctaagtct 124 target-01rs-12SNP-G12T-R agacttagatctgagccctccctcttcccagaacaggcaggggtaaaagc 125 HEX-N12-BHQ1 Hex-nnnnnnnnnnnn-Bhq1 126 FAM-N12-Eclipse Fam-nnnnnnnnnnnn-Eclipse 126

Table 2. Target sequences used for single guide RNA transcription

Name of the oligonucleotide Sequence (5'–3') Id. Sequence No.: T7-sgRNA-F gaaattaatacgactcactataggg 128 T7-sgRNA-gyrB tcgcgcttgtcgcgcagacgaatgatctacaacagtagaaattccctatagtgagtcgtattaatttc 129 ZLsgRNA-DNMT1-3-R aacagacatggaccatcaggggtg 130 ZLsgRNA-DNMT1-3-14 catggaccatcagggtgccac 131 ZLsgRNA-DNMT1-3-15 acatggaccatcagggtgccac 132 ZLsgRNA-DNMT1-3-16 gacatggaccatcagggtgcc 133 ZLsgRNA-DNMT1-3-17 agacatggaccatcagggtgc 134 ZLsgRNA-DNMT1-3-18 cagacatggaccatcaggggtg 135 ZLsgRNA-DNMT1-3-19 acagacatggaccatcaggggtg 136 ZL-01-rs-16-R ccagcacaggcaggggggtgccacttctcagatttgagaag 137 ZL-02-rs-16-R cgttatactatgactggtgccacttctcagatttgagaag 138 ZL-03-rs-16-R atgattgaaatgtcctgtgccacttctcagatttgagaag 139 ZL-01rs-16-G12T-R ccagaacaggcagggggtgccacttctcagatttgagaag 140 ZL-02rs-16-T12C-R cgttgtactatgactggtgccacttctcagatttgagaag 141 ZL-03rs-16-T12C-R atgactgaaatgtcctgtgccacttctcagatttgagaag 142 ZL-sgRNA-05rs-16 aggtccaggagcaggggtgccacttctcagatttgagaag 143 ZL-sgRNA-05rs--G11A-16 aggtctaggagcaggggtgccacttctcagatttgagaag 144 ZL-sgRNA-41rs-16 gatgacgtagatcagtgtgccacttctcagatttgagaag 145 ZL-sgRNA-41rs-G11A-16 gatgatgtagatcagtgtgccacttctcagatttgagaag 146 ZL-gyrB-crRNA2-R atatcttctacttctccactgtgccacttctcagatttgagaag 147 ZL-sry-crRNA3-R tctagagaatcccagaatgcgtgccacttctcagatttgagaag 148 ZL-02rs-16-11SNP-R gcgttatactatgactgtgccacttctcagatttgagaag 149 ZL-02rs-16-(T12C)11SNP-R gcgttgtactatgactgtgccacttctcagatttgagaag 150 ZL-01rs-10SNP-18-R cttcccagcacaggcagggtgccacttctcagatttgagaag 151 ZL-01rs-10SNPG10T-18-R cttcccagaacaggcagggtgccacttctcagatttgagaag 152 ZL-01rs-12SNP-18-R tcccagcacaggcagggggtgccacttctcagatttgagaag 153 ZL-01rs-12SNPG12T-18-R tcccagaacaggcagggggtgccacttctcagatttgagaag 154 ZL-sgRNA-JEV-E453-R tgtgatccaagacattccccgtgccacttctcagatttgagaag 155 ZL-sgRNA-JEV-NS170-R ctcgtcagtgctctcctctcgtgccacttctcagatttgagaag 156 ZL-01rs-10SNP-19-R tcttcccagcacaggcagggtgccacttctcagatttgagaag 157 ZL-01rs-10SNPG10T-19-R tcttcccagaacaggcagggtgccacttctcagatttgagaag 158 ZL-01rs-12SNP-19-R ttcccagcacaggcagggggtgccacttctcagatttgagaag 159 ZL-01rs-12SNPG12T-19-R ttcccagaacaggcagggggtgccacttctcagatttgagaag 160

Table 3. Oligonucleotides as amplification primers

Name of the oligonucleotide Sequence (5'–3') Id. Sequence No.: LAMP-DNM-F3 gtgaacgttcccttagcact 161 LAMP-DNM-B3 ggggggcagaactagtcc 162 LAMP-DNM-FIP cgccacttgacaggcgagtaactgccacttattggggtcagc 163 LAMP-DNM-BIP gcgtgttccccagagtgacttagcagcttcctcctcctt 164 LAMP-DNM-LoopF aggaaacattaacgtactgatg 165 LAMP-DNM-LoopB ttccttttttttcccttcagc 166 LAMP-gyrB-F3 cgacggcaaagaagacca 167 LAMP-gyrB-B3 agcctgccaggtgagtac 168 LAMP-gyrB-FIP cgggtggatcggcgttttgttcactatgaaggcggcatca 169 LAMP-gyrB-BIP gtattggcgtcgaagtggcgttcgctgcggaatgttgttg 170 LAMP-gyrB-LoopF ttgttcagatattcaacgaacg 171 LAMP-gyrB-LoopB gtggaacgatggcttccagg 172 LAMP-sry-F3 tctctgtgcatggcctgta 173 LAMP-sry-B3 aacagtaaaggcaacgtcca 174 LAMP-sry-FIP gcagctgggataccagtggaagtgcctcctggaagaatgg 175 LAMP-sry-BIP tctctagagccatcttgcgcctgaagcgacccatgaacgc 176 LAMP-sry-LoopF tgcttactgaagccgaaaaatg 177 LAMP-sry-LoopB tgatcgcgagaccacacgatg 178 ASP-primer ggtttcggatgttacagcgt 179 ASP-rs5082-F caagcaccccacccgctcacccacctcctcctttg 180 ASP-rs5082-R ggtttcggatgttacagcgtgtgctggaagacttagatctgag 181 LAMP-rs5082-F3 gctggaaaggtcaagggac 182 LAMP-rs5082-B3 ggggtttgttgcacagtcc 183 LAMP-rs5082-FIP ggtagaagcaaaggcaggaggtttgcccaaggtcacacag 184 LAMP-rs5082-FIP-10PAM gaaagaagcaaaggcaggaggtttgcccaaggtcacacag 185 LAMP-rs5082-BIP ctgggaagagggaggggctcagtgttgccacactttcactgg 186 LAMP-rs5082-LoopF gtgagcgggtggggtgct 187 LAMP-rs5082-LoopB tctaagtcttccagcacgggatc 188 LAMP-E453-F3 tgacacagcctgggactt 189 LAMP-E453-B3 cacacctcctgtggctaag 190 LAMP-E453-FIP gagtgttctgaaggcaccaccagctccattggaggggtct 191 LAMP-E453-BIP acacaagggctaatgggtgccgccaaagcaattgatcggtc 192 LAMP-E453-LoopF ttggtgaacggcttttcctatg 193 LAMP-E453-LoopB tgctctggatgggcgtcaacg 194 LAMP-NS170-F3 gagacaaaggaatgccctga 195 LAMP-NS170-B3 gccctctcaagtttccatgt 196 LAMP-NS170-FIP cgggttgatgtgatgccaaagcgagcacagagcttggaaca 197 LAMP-NS170-BIP ggagcgatcataggtacggctggcgactctcaatccagtacg 198 LAMP-NS170-LoopF gaagtcttcgatttgcatg 199 LAMP-NS170-LoopB acatgtggcagtccatagtgac 200 GAPDH-LAMP-target3-FIP agggatctcgctcctggaagatcaccgtcaaggctgagaac 201 GAPDH-LAMP-target3-BIP cgatgctggcgctgagtacgaatgagccccagccttct 202 GAPDH-LAMP-target3-F3 tccacccatggcaaattcc 203 GAPDH-LAMP-target3-B3 agggggcagagatgatgac 204 GAPDH-LAMP-target3-LoopF tccattgatgacaagcttccc 205 GAPDH-LAMP-target3-LoopB tcgtggagtccactggcgtc 206 CDR1as-LAMP-FIP ccagatcttcggaaaatccacatctgtatttgatggaagacctt 207 CDR1as-LAMP-BIP agaccagtaattgctggaagacttgtcttccaagaagctcc 208 CDR1as-LAMP-F3 agatttttctggaagacatgg 209 CDR1as-LAMP-B3 atgtcttccggacaatcc 210 CDR1as-LAMP-LoopB tgctggaagacttgatttactgg 211 COL1A2-F ggaggcaccctagggccagggaaa 212 COL1A2-R gttactgcaagcagcaacaaagtcc 213 COL1A2(BSP)-F ggaggtatttagggggttagggaaa 214 COL1A2(BSP)-R attactacaaaaaaaaaaatcc 215 sgRNA-GAPDH-target3-7 ggcgtcttcaccaccatggagtgccacttctcagatttgagaag 216 sgRNA-CDR1as-target1-3 ctccaagtcttccagtaaatgtgccacttctcagatttgagaag 217 sgRNA-COL1A2m3-C12-17 ctaccgtaatactaaaagtgccacttctcagatttgagaag 127

All publications mentioned in the present invention are incorporated by reference, as if each individual document were cited as an example by reference in this application. It should be understood that after becoming familiar with the above teachings of the present invention, various changes and modifications may be made by those skilled in the art. These equivalent forms also fall within the scope of the appended claims.

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SEQUENCE LIST

<110> SHANGHAI TOLO BIOTECHNOLOGY COMPANY LIMITED

<120> Use of highly heat-resistant Cas protein, and method and reagent kit

to detect the target nucleic acid molecule

<130> 431117

<150> CN201810560284.8

<151> 2018-06-03

<160> 217

<170>PatentIn version 3.5

<210> 1

<211> 66

<212> DNA

<213> artificial sequence

<400> 1

tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60

ggatcc 66

<210> 2

<211> 66

<212> DNA

<213> artificial sequence

<400> 2

ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60

cagaaa 66

<210> 3

<211> 66

<212> DNA

<213> artificial sequence

<400> 3

ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60

cagaaa 66

<210> 4

<211> 66

<212> DNA

<213> artificial sequence

<400> 4

ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60

cagaaa 66

<210> 5

<211> 50

<212> DNA

<213> artificial sequence

<400> 5

aatgttccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 6

<211> 50

<212> DNA

<213> artificial sequence

<400> 6

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggaacatt 50

<210> 7

<211> 50

<212> DNA

<213> artificial sequence

<400> 7

aatgaaccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 8

<211> 50

<212> DNA

<213> artificial sequence

<400> 8

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggttcatt 50

<210> 9

<211> 50

<212> DNA

<213> artificial sequence

<400> 9

aatgatccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 10

<211> 50

<212> DNA

<213> artificial sequence

<400> 10

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggatcatt 50

<210> 11

<211> 50

<212> DNA

<213> artificial sequence

<400> 11

aatgtaccct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 12

<211> 50

<212> DNA

<213> artificial sequence

<400> 12

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggtacatt 50

<210> 13

<211> 50

<212> DNA

<213> artificial sequence

<400> 13

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggcccatt 50

<210> 14

<211> 50

<212> DNA

<213> artificial sequence

<400> 14

gtcacgccac ttgacaggcg agtaacagac atggaccatc agggggcatt 50

<210> 15

<211> 50

<212> DNA

<213> artificial sequence

<400> 15

aatgttcgct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 16

<211> 50

<212> DNA

<213> artificial sequence

<400> 16

gtcacgccac ttgacaggcg agtaacagac atggaccatc agcgaacatt 50

<210> 17

<211> 50

<212> DNA

<213> artificial sequence

<400> 17

aatgttccgt gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 18

<211> 50

<212> DNA

<213> artificial sequence

<400> 18

gtcacgccac ttgacaggcg agtaacagac atggaccatc acggaacatt 50

<210> 19

<211> 50

<212> DNA

<213> artificial sequence

<400> 19

aatgttccca gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 20

<211> 50

<212> DNA

<213> artificial sequence

<400> 20

gtcacgccac ttgacaggcg agtaacagac atggaccatc tgggaacatt 50

<210> 21

<211> 50

<212> DNA

<213> artificial sequence

<400> 21

aatgttccct catggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 22

<211> 50

<212> DNA

<213> artificial sequence

<400> 22

gtcacgccac ttgacaggcg agtaacagac atggaccatg agggaacatt 50

<210> 23

<211> 50

<212> DNA

<213> artificial sequence

<400> 23

aatgttccct gttggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 24

<211> 50

<212> DNA

<213> artificial sequence

<400> 24

gtcacgccac ttgacaggcg agtaacagac atggaccaac agggaacatt 50

<210> 25

<211> 50

<212> DNA

<213> artificial sequence

<400> 25

aatgttccct gaaggtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 26

<211> 50

<212> DNA

<213> artificial sequence

<400> 26

gtcacgccac ttgacaggcg agtaacagac atggaccttc agggaacatt 50

<210> 27

<211> 50

<212> DNA

<213> artificial sequence

<400> 27

aatgttccct gatcgtccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 28

<211> 50

<212> DNA

<213> artificial sequence

<400> 28

gtcacgccac ttgacaggcg agtaacagac atggacgatc agggaacatt 50

<210> 29

<211> 50

<212> DNA

<213> artificial sequence

<400> 29

aatgttccct gatgctccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 30

<211> 50

<212> DNA

<213> artificial sequence

<400> 30

gtcacgccac ttgacaggcg agtaacagac atggagcatc agggaacatt 50

<210> 31

<211> 50

<212> DNA

<213> artificial sequence

<400> 31

aatgttccct gatggaccat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 32

<211> 50

<212> DNA

<213> artificial sequence

<400> 32

gtcacgccac ttgacaggcg agtaacagac atggtccatc agggaacatt 50

<210> 33

<211> 50

<212> DNA

<213> artificial sequence

<400> 33

aatgttccct gatggtgcat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 34

<211> 50

<212> DNA

<213> artificial sequence

<400> 34

gtcacgccac ttgacaggcg agtaacagac atgcaccatc agggaacatt 50

<210> 35

<211> 50

<212> DNA

<213> artificial sequence

<400> 35

aatgttccct gatggtcgat gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 36

<211> 50

<212> DNA

<213> artificial sequence

<400> 36

gtcacgccac ttgacaggcg agtaacagac atcgaccatc agggaacatt 50

<210> 37

<211> 50

<212> DNA

<213> artificial sequence

<400> 37

aatgttccct gatggtcctt gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 38

<211> 50

<212> DNA

<213> artificial sequence

<400> 38

gtcacgccac ttgacaggcg agtaacagac aaggaccatc agggaacatt 50

<210> 39

<211> 50

<212> DNA

<213> artificial sequence

<400> 39

aatgttccct gatggtccaa gtctgttact cgcctgtcaa gtggcgtgac 50

<210> 40

<211> 50

<212> DNA

<213> artificial sequence

<400> 40

gtcacgccac ttgacaggcg agtaacagac ttggaccatc agggaacatt 50

<210> 41

<211> 50

<212> DNA

<213> artificial sequence

<400> 41

aatgttccct gatggtccat ctctgttact cgcctgtcaa gtggcgtgac 50

<210> 42

<211> 50

<212> DNA

<213> artificial sequence

<400> 42

gtcacgccac ttgacaggcg agtaacagag atggaccatc agggaacatt 50

<210> 43

<211> 50

<212> DNA

<213> artificial sequence

<400> 43

aatgttccct gatggtccat gactgttact cgcctgtcaa gtggcgtgac 50

<210> 44

<211> 50

<212> DNA

<213> artificial sequence

<400> 44

gtcacgccac ttgacaggcg agtaacagtc atggaccatc agggaacatt 50

<210> 45

<211> 50

<212> DNA

<213> artificial sequence

<400> 45

aatgttccct gatggtccat gtgtgttact cgcctgtcaa gtggcgtgac 50

<210> 46

<211> 50

<212> DNA

<213> artificial sequence

<400> 46

gtcacgccac ttgacaggcg agtaacacac atggaccatc agggaacatt 50

<210> 47

<211> 50

<212> DNA

<213> artificial sequence

<400> 47

aatgttccct gatggtccat gtcagttact cgcctgtcaa gtggcgtgac 50

<210> 48

<211> 50

<212> DNA

<213> artificial sequence

<400> 48

gtcacgccac ttgacaggcg agtaactgac atggaccatc agggaacatt 50

<210> 49

<211> 50

<212> DNA

<213> artificial sequence

<400> 49

agacttagat ctgagccctc cctcttccca gcacaggcag gggtagaagc 50

<210> 50

<211> 50

<212> DNA

<213> artificial sequence

<400> 50

agacttagat ctgagccctc cctcttccca gaacaggcag gggtagaagc 50

<210> 51

<211> 50

<212> DNA

<213> artificial sequence

<400> 51

tgtgtttgga tttgcagtag gctgaagcgt tatactatga ctggagtcca 50

<210> 52

<211> 50

<212> DNA

<213> artificial sequence

<400> 52

tgtgtttgga tttgcagtag gctgaagcgt tgtactatga ctggagtcca 50

<210> 53

<211> 50

<212> DNA

<213> artificial sequence

<400> 53

aaaatagtgc tttttacttt tatctgaatg attgaaatgt ccttttccca 50

<210> 54

<211> 50

<212> DNA

<213> artificial sequence

<400> 54

aaaatagtgc tttttacttt tatctgaatg actgaaatgt ccttttccca 50

<210> 55

<211> 50

<212> DNA

<213> artificial sequence

<400> 55

gtcacgccac ttgacaggcg agtaacagac atggaccatc agtgaacatt 50

<210> 56

<211> 50

<212> DNA

<213> artificial sequence

<400> 56

gtcacgccac ttgacaggcg agtaacagac atggaccatc agagaacatt 50

<210> 57

<211> 50

<212> DNA

<213> artificial sequence

<400> 57

gtcacgccac ttgacaggcg agtaacagac atggaccatc aaggaacatt 50

<210> 58

<211> 50

<212> DNA

<213> artificial sequence

<400> 58

gtcacgccac ttgacaggcg agtaacagac atggaccatc atggaacatt 50

<210> 59

<211> 50

<212> DNA

<213> artificial sequence

<400> 59

gtcacgccac ttgacaggcg agtaacagac atggaccatc cgggaacatt 50

<210> 60

<211> 50

<212> DNA

<213> artificial sequence

<400> 60

gtcacgccac ttgacaggcg agtaacagac atggaccatc ggggaacatt 50

<210> 61

<211> 50

<212> DNA

<213> artificial sequence

<400> 61

gtcacgccac ttgacaggcg agtaacagac atggaccata agggaacatt 50

<210> 62

<211> 50

<212> DNA

<213> artificial sequence

<400> 62

gtcacgccac ttgacaggcg agtaacagac atggaccatt agggaacatt 50

<210> 63

<211> 50

<212> DNA

<213> artificial sequence

<400> 63

gtcacgccac ttgacaggcg agtaacagac atggaccacc agggaacatt 50

<210> 64

<211> 50

<212> DNA

<213> artificial sequence

<400> 64

gtcacgccac ttgacaggcg agtaacagac atggaccagc agggaacatt 50

<210> 65

<211> 50

<212> DNA

<213> artificial sequence

<400> 65

gtcacgccac ttgacaggcg agtaacagac atggaccctc agggaacatt 50

<210> 66

<211> 50

<212> DNA

<213> artificial sequence

<400> 66

gtcacgccac ttgacaggcg agtaacagac atggaccgtc agggaacatt 50

<210> 67

<211> 50

<212> DNA

<213> artificial sequence

<400> 67

gtcacgccac ttgacaggcg agtaacagac atggacaatc agggaacatt 50

<210> 68

<211> 50

<212> DNA

<213> artificial sequence

<400> 68

gtcacgccac ttgacaggcg agtaacagac atggactatc agggaacatt 50

<210> 69

<211> 50

<212> DNA

<213> artificial sequence

<400> 69

gtcacgccac ttgacaggcg agtaacagac atggaacatc agggaacatt 50

<210> 70

<211> 50

<212> DNA

<213> artificial sequence

<400> 70

gtcacgccac ttgacaggcg agtaacagac atggatcatc agggaacatt 50

<210> 71

<211> 50

<212> DNA

<213> artificial sequence

<400> 71

gtcacgccac ttgacaggcg agtaacagac atggcccatc agggaacatt 50

<210> 72

<211> 50

<212> DNA

<213> artificial sequence

<400> 72

gtcacgccac ttgacaggcg agtaacagac atgggccatc agggaacatt 50

<210> 73

<211> 50

<212> DNA

<213> artificial sequence

<400> 73

gtcacgccac ttgacaggcg agtaacagac atgaaccatc agggaacatt 50

<210> 74

<211> 50

<212> DNA

<213> artificial sequence

<400> 74

gtcacgccac ttgacaggcg agtaacagac atgtaccatc agggaacatt 50

<210> 75

<211> 50

<212> DNA

<213> artificial sequence

<400> 75

gtcacgccac ttgacaggcg agtaacagac atagaccatc agggaacatt 50

<210> 76

<211> 50

<212> DNA

<213> artificial sequence

<400> 76

gtcacgccac ttgacaggcg agtaacagac attgaccatc agggaacatt 50

<210> 77

<211> 50

<212> DNA

<213> artificial sequence

<400> 77

gtcacgccac ttgacaggcg agtaacagac acggaccatc agggaacatt 50

<210> 78

<211> 50

<212> DNA

<213> artificial sequence

<400> 78

gtcacgccac ttgacaggcg agtaacagac agggaccatc agggaacatt 50

<210> 79

<211> 50

<212> DNA

<213> artificial sequence

<400> 79

gtcacgccac ttgacaggcg agtaacagac ctggaccatc agggaacatt 50

<210> 80

<211> 50

<212> DNA

<213> artificial sequence

<400> 80

gtcacgccac ttgacaggcg agtaacagac gtggaccatc agggaacatt 50

<210> 81

<211> 50

<212> DNA

<213> artificial sequence

<400> 81

gtcacgccac ttgacaggcg agtaacagaa atggaccatc agggaacatt 50

<210> 82

<211> 50

<212> DNA

<213> artificial sequence

<400> 82

gtcacgccac ttgacaggcg agtaacagat atggaccatc agggaacatt 50

<210> 83

<211> 50

<212> DNA

<213> artificial sequence

<400> 83

gtcacgccac ttgacaggcg agtaacagcc atggaccatc agggaacatt 50

<210> 84

<211> 50

<212> DNA

<213> artificial sequence

<400> 84

gtcacgccac ttgacaggcg agtaacaggc atggaccatc agggaacatt 50

<210> 85

<211> 50

<212> DNA

<213> artificial sequence

<400> 85

gtcacgccac ttgacaggcg agtaacaaac atggaccatc agggaacatt 50

<210> 86

<211> 50

<212> DNA

<213> artificial sequence

<400> 86

gtcacgccac ttgacaggcg agtaacatac atggaccatc agggaacatt 50

<210> 87

<211> 50

<212> DNA

<213> artificial sequence

<400> 87

agacttagat ctgagccctc cctcttccca gcacacgcag gggtagaagc 50

<210> 88

<211> 50

<212> DNA

<213> artificial sequence

<400> 88

agacttagat ctgagccctc cctcttccca gcactggcag gggtagaagc 50

<210> 89

<211> 50

<212> DNA

<213> artificial sequence

<400> 89

agacttagat ctgagccctc cctcttccca gcagaggcag gggtagaagc 50

<210> 90

<211> 50

<212> DNA

<213> artificial sequence

<400> 90

agacttagat ctgagccctc cctcttccca gctcaggcag gggtagaagc 50

<210> 91

<211> 50

<212> DNA

<213> artificial sequence

<400> 91

agacttagat ctgagccctc cctcttccca ggacaggcag gggtagaagc 50

<210> 92

<211> 50

<212> DNA

<213> artificial sequence

<400> 92

agacttagat ctgagccctc cctcttccca ccacaggcag gggtagaagc 50

<210> 93

<211> 50

<212> DNA

<213> artificial sequence

<400> 93

agacttagat ctgagccctc cccttccct gcacaggcag gggtagaagc 50

<210> 94

<211> 50

<212> DNA

<213> artificial sequence

<400> 94

agacttagat ctgagccctc cccttccga gcacaggcag gggtagaagc 50

<210> 95

<211> 50

<212> DNA

<213> artificial sequence

<400> 95

agacttagat ctgagccctc cctcttcgca gcacaggcag gggtagaagc 50

<210> 96

<211> 50

<212> DNA

<213> artificial sequence

<400> 96

tgtgtttgga tttgcagtag gctgaagcgt tatacaatga ctggagtcca 50

<210> 97

<211> 50

<212> DNA

<213> artificial sequence

<400> 97

tgtgtttgga tttgcagtag gctgaagcgt tatagtatga ctggagtcca 50

<210> 98

<211> 50

<212> DNA

<213> artificial sequence

<400> 98

tgtgtttgga tttgcagtag gctgaagcgt tattctatga ctggagtcca 50

<210> 99

<211> 50

<212> DNA

<213> artificial sequence

<400> 99

tgtgtttgga tttgcagtag gctgaagcgt taaactatga ctggagtcca 50

<210> 100

<211> 50

<212> DNA

<213> artificial sequence

<400> 100

tgtgtttgga tttgcagtag gctgaagcgt tttactatga ctggagtcca 50

<210> 101

<211> 50

<212> DNA

<213> artificial sequence

<400> 101

tgtgtttgga tttgcagtag gctgaagcgt aatactatga ctggagtcca 50

<210> 102

<211> 50

<212> DNA

<213> artificial sequence

<400> 102

tgtgtttgga tttgcagtag gctgaagcga tatactatga ctggagtcca 50

<210> 103

<211> 50

<212> DNA

<213> artificial sequence

<400> 103

tgtgtttgga tttgcagtag gctgaagcct tatactatga ctggagtcca 50

<210> 104

<211> 50

<212> DNA

<213> artificial sequence

<400> 104

tgtgtttgga tttgcagtag gctgaagggt tatactatga ctggagtcca 50

<210> 105

<211> 50

<212> DNA

<213> artificial sequence

<400> 105

aaaatagtgc tttttacttt tatctgaatg attgatatgt ccttttccca 50

<210> 106

<211> 50

<212> DNA

<213> artificial sequence

<400> 106

aaaatagtgc tttttacttt tatctgaatg attgtaatgt ccttttccca 50

<210> 107

<211> 50

<212> DNA

<213> artificial sequence

<400> 107

aaaatagtgc tttttacttt tatctgaatg attcaaatgt ccttttccca 50

<210> 108

<211> 50

<212> DNA

<213> artificial sequence

<400> 108

aaaatagtgc tttttacttt tatctgaatg atagaaatgt ccttttccca 50

<210> 109

<211> 50

<212> DNA

<213> artificial sequence

<400> 109

aaaaatgtgc tttttacttt tatctgaatg aatgaaatgt ccttttccca 50

<210> 110

<211> 50

<212> DNA

<213> artificial sequence

<400> 110

aaaatagtgc tttttacttt tatctgaatg tttgaaatgt ccttttccca 50

<210> 111

<211> 50

<212> DNA

<213> artificial sequence

<400> 111

aaaatagtgc tttttacttt tatctgaatc attgaaatgt ccttttccca 50

<210> 112

<211> 50

<212> DNA

<213> artificial sequence

<400> 112

aaaatagtgc tttttacttt tatctgaaag attgaaatgt ccttttccca 50

<210> 113

<211> 50

<212> DNA

<213> artificial sequence

<400> 113

aaaatagtgc tttttacttt tatctgattg attgaaatgt ccttttccca 50

<210> 114

<211> 50

<212> DNA

<213> artificial sequence

<400> 114

ggatctcctg gcggaggtgg tggtagaagg tccaggagca ggggtagccg 50

<210> 115

<211> 50

<212> DNA

<213> artificial sequence

<400> 115

ggatctcctg gcggaggtgg tggtagaagg tctaggagca ggggtagccg 50

<210> 116

<211> 50

<212> DNA

<213> artificial sequence

<400> 116

agcacatgtt tgttgaatga gtcagtggat gacgtagatc agtatcttgt 50

<210> 117

<211> 50

<212> DNA

<213> artificial sequence

<400> 117

agcacatgtt tgttgaatga gtcagtggat gatgtagatc agtatcttgt 50

<210> 118

<211> 50

<212> DNA

<213> artificial sequence

<400> 118

gcttctttcc ctgcctgtgc tgggaagagg gagggctcag atctaagtct 50

<210> 119

<211> 50

<212> DNA

<213> artificial sequence

<400> 119

agacttagat ctgagccctc cctcttccca gcacaggcag ggaaagaagc 50

<210> 120

<211> 50

<212> DNA

<213> artificial sequence

<400> 120

gcttctttcc ctgcctgttc tgggaagagg gagggctcag atctaagtct 50

<210> 121

<211> 50

<212> DNA

<213> artificial sequence

<400> 121

agacttagat ctgagccctc cctcttccca gaacaggcag ggaaagaagc 50

<210> 122

<211> 50

<212> DNA

<213> artificial sequence

<400> 122

gcttttaccc ctgcctgtgc tgggaagagg gagggctcag atctaagtct 50

<210> 123

<211> 50

<212> DNA

<213> artificial sequence

<400> 123

agacttagat ctgagccctc cctcttccca gcacaggcag gggtaaaagc 50

<210> 124

<211> 50

<212> DNA

<213> artificial sequence

<400> 124

gcttttaccc ctgcctgttc tgggaagagg gagggctcag atctaagtct 50

<210> 125

<211> 50

<212> DNA

<213> artificial sequence

<400> 125

agacttagat ctgagccctc cctcttccca gaacaggcag gggtaaaagc 50

<210> 126

<211> 12

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (1)..(12)

<223> n is a, c, g, or t

<400> 126

nnnnnnnnnn nn 12

<210> 127

<211> 41

<212> DNA

<213> artificial sequence

<400> 127

ctaccgtaat actaaaagtg ccacttctca gatttgagaa g 41

<210> 128

<211> 25

<212> DNA

<213> artificial sequence

<400> 128

gaaattaata cgactcacta taggg 25

<210> 129

<211> 68

<212> DNA

<213> artificial sequence

<400> 129

tcgcgcttgt cgcgcagacg aatgatctac aacagtagaa attccctata gtgagtcgta 60

ttaatttc 68

<210> 130

<211> 23

<212> DNA

<213> artificial sequence

<400> 130

aacagacatg gaccatcagg gtg 23

<210> 131

<211> 21

<212> DNA

<213> artificial sequence

<400> 131

catggaccat cagggtgcca c 21

<210> 132

<211> 22

<212> DNA

<213> artificial sequence

<400> 132

acatggacca tcagggtgcc ac 22

<210> 133

<211> 21

<212> DNA

<213> artificial sequence

<400> 133

gacatggacc atcaggggtgc c 21

<210> 134

<211> 21

<212> DNA

<213> artificial sequence

<400> 134

agacatggac catcaggggtg c 21

<210> 135

<211> 21

<212> DNA

<213> artificial sequence

<400> 135

cagacatgga ccatcagggt g 21

<210> 136

<211> 22

<212> DNA

<213> artificial sequence

<400> 136

acagacatgg accatcaggg tg 22

<210> 137

<211> 40

<212> DNA

<213> artificial sequence

<400> 137

ccagcacagg caggggggtgc cacttctcag atttgagaag 40

<210> 138

<211> 40

<212> DNA

<213> artificial sequence

<400> 138

cgttatacta tgactggtgc cacttctcag atttgagaag 40

<210> 139

<211> 40

<212> DNA

<213> artificial sequence

<400> 139

atgattgaaa tgtcctgtgc cacttctcag atttgagaag 40

<210> 140

<211> 40

<212> DNA

<213> artificial sequence

<400> 140

ccagaacagg cagggggtgc cacttctcag atttgagaag 40

<210> 141

<211> 40

<212> DNA

<213> artificial sequence

<400> 141

cgttgtacta tgactggtgc cacttctcag atttgagaag 40

<210> 142

<211> 40

<212> DNA

<213> artificial sequence

<400> 142

atgactgaaa tgtcctgtgc cacttctcag atttgagaag 40

<210> 143

<211> 40

<212> DNA

<213> artificial sequence

<400> 143

aggtccagga gcaggggtgc cacttctcag atttgagaag 40

<210> 144

<211> 40

<212> DNA

<213> artificial sequence

<400> 144

aggtctagga gcaggggtgc cacttctcag atttgagaag 40

<210> 145

<211> 40

<212> DNA

<213> artificial sequence

<400> 145

gatgacgtag atcagtgtgc cacttctcag atttgagaag 40

<210> 146

<211> 40

<212> DNA

<213> artificial sequence

<400> 146

gatgatgtag atcagtgtgc cacttctcag atttgagaag 40

<210> 147

<211> 44

<212> DNA

<213> artificial sequence

<400> 147

atatcttcta cttctccact gtgccacttc tcagatttga gaag 44

<210> 148

<211> 44

<212> DNA

<213> artificial sequence

<400> 148

tctagagaat cccagaatgc gtgccacttc tcagatttga gaag 44

<210> 149

<211> 40

<212> DNA

<213> artificial sequence

<400> 149

gcgttatact atgactgtgc cacttctcag atttgagaag 40

<210> 150

<211> 40

<212> DNA

<213> artificial sequence

<400> 150

gcgttgtact atgactgtgc cacttctcag atttgagaag 40

<210> 151

<211> 42

<212> DNA

<213> artificial sequence

<400> 151

cttcccagca caggcagggt gccacttctc agatttgaga ag 42

<210> 152

<211> 42

<212> DNA

<213> artificial sequence

<400> 152

cttcccagaa caggcagggt gccacttctc agatttgaga ag 42

<210> 153

<211> 42

<212> DNA

<213> artificial sequence

<400> 153

tcccagcaca ggcagggggt gccacttctc agatttgaga ag 42

<210> 154

<211> 42

<212> DNA

<213> artificial sequence

<400> 154

tcccagaaca ggcagggggt gccacttctc agatttgaga ag 42

<210> 155

<211> 44

<212> DNA

<213> artificial sequence

<400> 155

tgtgatccaa gacattcccc gtgccacttc tcagatttga gaag 44

<210> 156

<211> 44

<212> DNA

<213> artificial sequence

<400> 156

ctcgtcagtg ctctcctctc gtgccacttc tcagatttga gaag 44

<210> 157

<211> 43

<212> DNA

<213> artificial sequence

<400> 157

tcttcccagc acaggcaggg tgccacttct cagatttgag aag 43

<210> 158

<211> 43

<212> DNA

<213> artificial sequence

<400> 158

tcttcccaga acaggcaggg tgccacttct cagatttgag aag 43

<210> 159

<211> 43

<212> DNA

<213> artificial sequence

<400> 159

ttcccagcac aggcaggggg tgccacttct cagatttgag aag 43

<210> 160

<211> 43

<212> DNA

<213> artificial sequence

<400> 160

ttcccagaac aggcaggggg tgccacttct cagatttgag aag 43

<210> 161

<211> 20

<212> DNA

<213> artificial sequence

<400> 161

gtgaacgttc ccttagcact 20

<210> 162

<211> 19

<212> DNA

<213> artificial sequence

<400> 162

gggaggggcag aactagtcc 19

<210> 163

<211> 41

<212> DNA

<213> artificial sequence

<400> 163

cgccacttga caggcgagta actgccactt attgggtcag c 41

<210> 164

<211> 39

<212> DNA

<213> artificial sequence

<400> 164

gcgtgttccc cagagtgact tagcagcttc ctcctcctt 39

<210> 165

<211> 22

<212> DNA

<213> artificial sequence

<400> 165

aggaaacatt aacgtactga tg 22

<210> 166

<211> 21

<212> DNA

<213> artificial sequence

<400> 166

ttccttttat ttcccttcag c 21

<210> 167

<211> 18

<212> DNA

<213> artificial sequence

<400> 167

cgacggcaaa gaagacca 18

<210> 168

<211> 18

<212> DNA

<213> artificial sequence

<400> 168

agcctgccag gtgagtac 18

<210> 169

<211> 40

<212> DNA

<213> artificial sequence

<400> 169

cgggtggatc ggcgttttgt tcactatgaa ggcggcatca 40

<210> 170

<211> 40

<212> DNA

<213> artificial sequence

<400> 170

gtattggcgt cgaagtggcg ttcgctgcgg aatgttgttg 40

<210> 171

<211> 22

<212> DNA

<213> artificial sequence

<400> 171

ttgttcagat attcaacgaa cg 22

<210> 172

<211> 20

<212> DNA

<213> artificial sequence

<400> 172

gtggaacgat ggcttccagg 20

<210> 173

<211> 19

<212> DNA

<213> artificial sequence

<400> 173

tctctgtgca tggcctgta 19

<210> 174

<211> 20

<212> DNA

<213> artificial sequence

<400> 174

aacagtaaag gcaacgtcca 20

<210> 175

<211> 40

<212> DNA

<213> artificial sequence

<400> 175

gcagctggga taccagtgga agtgcctcct ggaagaatgg 40

<210> 176

<211> 40

<212> DNA

<213> artificial sequence

<400> 176

tctctagagc catcttgcgc ctgaagcgac ccatgaacgc 40

<210> 177

<211> 22

<212> DNA

<213> artificial sequence

<400> 177

tgcttactga agccgaaaaa tg 22

<210> 178

<211> 21

<212> DNA

<213> artificial sequence

<400> 178

tgatcgcgag accacacgat g 21

<210> 179

<211> 20

<212> DNA

<213> artificial sequence

<400> 179

ggtttcggat gttacagcgt 20

<210> 180

<211> 35

<212> DNA

<213> artificial sequence

<400> 180

caagcacccc acccgctcac ccacctcctc ctttg 35

<210> 181

<211> 43

<212> DNA

<213> artificial sequence

<400> 181

gtttcggat gttacagcgt gtgctggaag acttagatct gag 43

<210> 182

<211> 19

<212> DNA

<213> artificial sequence

<400> 182

gctggaaagg tcaagggac 19

<210> 183

<211> 19

<212> DNA

<213> artificial sequence

<400> 183

ggggtttgtt gcacagtcc 19

<210> 184

<211> 40

<212> DNA

<213> artificial sequence

<400> 184

ggtagaagca aaggcaggag gtttgcccaa ggtcacacag 40

<210> 185

<211> 40

<212> DNA

<213> artificial sequence

<400> 185

gaaagaagca aaggcaggag gtttgcccaa ggtcacacag 40

<210> 186

<211> 41

<212> DNA

<213> artificial sequence

<400> 186

ctgggaagag ggagggctca gtgttgccac actttcactg g 41

<210> 187

<211> 18

<212> DNA

<213> artificial sequence

<400> 187

gtgagcgggt ggggtgct 18

<210> 188

<211> 23

<212> DNA

<213> artificial sequence

<400> 188

tctaagtctt ccagcacgggg atc 23

<210> 189

<211> 18

<212> DNA

<213> artificial sequence

<400> 189

tgacacagcc tgggactt 18

<210> 190

<211> 19

<212> DNA

<213> artificial sequence

<400> 190

cacacctcct gtggctaag 19

<210> 191

<211> 40

<212> DNA

<213> artificial sequence

<400> 191

gagtgttctg aaggcaccac cagctccatt ggaggggtct 40

<210> 192

<211> 41

<212> DNA

<213> artificial sequence

<400> 192

acacaagggc taatggggtgc cgccaaagca attgatcggt c 41

<210> 193

<211> 22

<212> DNA

<213> artificial sequence

<400> 193

ttggtgaacg gcttttccta tg 22

<210> 194

<211> 21

<212> DNA

<213> artificial sequence

<400> 194

tgctctggat gggcgtcaac g 21

<210> 195

<211> 20

<212> DNA

<213> artificial sequence

<400> 195

gagacaaagg aatgccctga 20

<210> 196

<211> 20

<212> DNA

<213> artificial sequence

<400> 196

gccctctcaa gtttccatgt 20

<210> 197

<211> 41

<212> DNA

<213> artificial sequence

<400> 197

cgggttgatg tgatgccaaa gcgagcacag agcttggaac a 41

<210> 198

<211> 42

<212> DNA

<213> artificial sequence

<400> 198

ggagcgatca taggtacggc tggcgactct caatccagta cg 42

<210> 199

<211> 19

<212> DNA

<213> artificial sequence

<400> 199

gaagtcttcg atttgcatg 19

<210> 200

<211> 22

<212> DNA

<213> artificial sequence

<400> 200

acatgtggca gtccatagtg ac 22

<210> 201

<211> 41

<212> DNA

<213> artificial sequence

<400> 201

agggatctcg ctcctggaag atcaccgtca aggctgagaa c 41

<210> 202

<211> 38

<212> DNA

<213> artificial sequence

<400> 202

cgatgctggc gctgagtacg aatgagcccc agccttct 38

<210> 203

<211> 19

<212> DNA

<213> artificial sequence

<400> 203

tccacccatg gcaaattcc 19

<210> 204

<211> 19

<212> DNA

<213> artificial sequence

<400> 204

agggggcaga gatgatgac 19

<210> 205

<211> 21

<212> DNA

<213> artificial sequence

<400> 205

tccattgatg acaagcttcc c 21

<210> 206

<211> 20

<212> DNA

<213> artificial sequence

<400> 206

tcgtggagtc cactggcgtc 20

<210> 207

<211> 46

<212> DNA

<213> artificial sequence

<400> 207

ccagatcttc caggaaaatc cacatctgta tttgatggaa gacctt 46

<210> 208

<211> 41

<212> DNA

<213> artificial sequence

<400> 208

agaccagtaa ttgctggaag acttgtcttc caagaagctc c 41

<210> 209

<211> 21

<212> DNA

<213> artificial sequence

<400> 209

agatttttct ggaagacatg g 21

<210> 210

<211> 18

<212> DNA

<213> artificial sequence

<400> 210

atgtcttccg gacaatcc 18

<210> 211

<211> 23

<212> DNA

<213> artificial sequence

<400> 211

tgctggaaga cttgatttac tgg 23

<210> 212

<211> 24

<212> DNA

<213> artificial sequence

<400> 212

ggaggcaccc tagggccagg gaaa 24

<210> 213

<211> 25

<212> DNA

<213> artificial sequence

<400> 213

gttactgcaa gcagcaacaa agtcc 25

<210> 214

<211> 24

<212> DNA

<213> artificial sequence

<400> 214

ggaggtattt tagggttaggg gaaa 24

<210> 215

<211> 25

<212> DNA

<213> artificial sequence

<400> 215

attactacaa acaacaacaa aatcc 25

<210> 216

<211> 44

<212> DNA

<213> artificial sequence

<400> 216

ggcgtcttca ccaccatgga gtgccacttc tcagatttga gaag 44

<210> 217

<211> 44

<212> DNA

<213> artificial sequence

<400> 217

ctccaagtct tccagtaaat gtgccacttc tcagatttga gaag 44

<---

Claims (82)

1. A detection system for detecting target nucleic acid molecules, the system comprising: (a) Cas12b protein; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; And (c) a nucleic acid probe that is single stranded DNA; wherein the target nucleic acid molecule is a target DNA. 2. The detection system according to claim 1, wherein the detection system includes a nucleic acid amplification reaction and a bypass digestion reaction, wherein the nucleic acid amplification reaction and the bypass digestion reaction are performed simultaneously, and the detection system is a one-step reaction system. 3. The detection system according to claim 1, wherein the Cas12b protein is selected from the group consisting of: AacCas12b ( Alicyclobacillus acidoterrestris ), Aac2Cas12b ( Alicyclobacillus acidiphilus ), AkaCas12b ( Alicyclobacillus kakegawensis ), AmaCas12b ( Alicyclobacillus macrosporangiidus ), AheCas12b ( Alicyclobac illus herbarius ) and AcoCas12b ( Alicyclobacillus contaminans ). 4. The detection system of claim 1, wherein when the target nucleic acid molecule is single-stranded DNA, the detection site of the target nucleic acid molecule is at position 9 or at positions 10-16, preferably at positions 10-14 and more preferably at positions 10 –12 behind the PAM guide RNA sequence. 5. The detection system of claim 4, wherein when the detection site is at position 9 behind the PAM sequence of the guide RNA, the detection site is G. 6. The detection system of claim 4 wherein the detection site of the target nucleic acid molecule is at positions 1-12, preferably at position 1, 3 or 10, downstream of the PAM sequence of the guide RNA. 7. SNP detection system (single nucleotide polymorphisms), while this system contains the first detection system and the second detection system; wherein the first detection system includes: (a1) Cas12b protein; (b1) a first guide RNA, wherein the first guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; And (c1) a nucleic acid probe that is single stranded DNA; and the second detection system includes: (a2) Cas12b protein; (b2) a second guide RNA, wherein the second guide RNA guides the Cas12b protein for specific binding to target nucleic acid molecules; And (c2) a nucleic acid probe that is single stranded DNA; wherein the target nucleic acid molecule is a target DNA; in addition, the first guide RNA and the second guide RNA target the same region of the nucleic acid sequence containing the SNP site, and the first guide RNA targets the wild-type or unmutated SNP site nucleic acid sequence, and the second guide RNA targets the sequence mutated nucleic acid SNP site. 8. The detection system according to claim 7, wherein the detection system includes a nucleic acid amplification reaction and a bypass cleavage reaction, wherein the nucleic acid amplification reaction and the bypass cleavage reaction are performed simultaneously, and the detection system is a one-step reaction system. 9. The detection system of claim 7, which further comprises a blank control system, and the blank control system includes: (a3) Cas12b protein; (c3) a nucleic acid probe that is single stranded DNA. 10. Kit for the detection of target nucleic acid molecules, which includes: i) the first container and the Cas12b protein found in the first container; ii) a second container and a guide RNA located in the second container, the second guide RNA directing the Cas12b protein for specific binding to target nucleic acid molecules; iii) a third container and a nucleic acid probe contained in the third container; iv) optionally a fourth container and a buffer located in the fourth container; wherein the target nucleic acid molecule is a target DNA. 11. The kit according to claim 10, which additionally contains: v) a fifth container and a polymerase for amplifying the target DNA contained in the fifth container; vi) an optional sixth container and reverse transcriptase for reverse transcription, located in the sixth container; vii) a seventh container and a dNTP for the amplification and/or reverse transcription reaction contained in the seventh container. 12. The kit of claim 11 wherein the polymerase is Bst 3.0 DNA polymerase. 13. A method for detecting the presence of target nucleic acid molecules in a sample, which includes the following steps: (i) providing a system for detecting target nucleic acid molecules according to claim 1, and a sample to be detected; and adding the sample to the detection system; And (ii) ascertaining whether the nucleic acid probe is cleaved in the Cas12b protein detection system, the cleavage being a trans cleavage of a bypass single-stranded DNA; however, if the nucleic acid probe is cleaved by the Cas12b protein, this indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas12b protein, this indicates the absence of the target nucleic acid molecule in the sample. 14. A method for detecting the presence of target nucleic acid molecules in a sample, which includes the following steps: (i) providing the detection system and sample for testing, and adding the sample to the detection system, while the detection system includes: (a) Cas12b protein; (b) a guide RNA, wherein the guide RNA guides the Cas12b protein to specifically bind to target nucleic acid molecules; (c) a nucleic acid probe that is single stranded DNA; wherein the target nucleic acid molecule is a target DNA; (d) buffer; (e1) a polymerase that is used to amplify the target DNA; (e2) an optional reverse transcriptase which is used for reverse transcription; And (e3) dNTP, which is used for the amplification reaction and/or the reverse transcription reaction; (ii) performing a reverse transcription and/or amplification reaction on the detection system, thereby obtaining a reverse transcription and/or amplified detection system; (iii) ascertaining whether the nucleic acid probe in the detection system obtained in the previous step is cleaved by the Cas12b protein, the cleavage being a trans cleavage of a bypass single-stranded DNA; however, if the nucleic acid probe is cleaved by the Cas12b protein, this indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas12b protein, this indicates the absence of the target nucleic acid molecule in the sample. 15. The method of claim 14, wherein the detection system includes a nucleic acid amplification reaction and a bypass digestion reaction, wherein the nucleic acid amplification reaction and the bypass digestion reaction are performed simultaneously, and the detection system is a one-step reaction system. 16. The method according to p. 14, in which the detection contains: qualitative detection or quantitative detection; in a preferred embodiment, the quantitative detection is an absolute quantitative detection; more preferably, the assay is assay combined with digital PCR technology. 17. The method of claim 14, wherein the nucleic acid amplification method is selected from the group consisting of: PCR amplification, loop isothermal amplification (LAMP), recombinase polymerase amplification (RPA), ligase chain reaction, branched DNA amplification, amplification, Nucleic acid sequence-based (NASBA), strand displacement amplification (SDA), transcription-mediated amplification, rolling ring amplification, helicase-dependent amplification (HDA), single primer isothermal amplification (SPIA), nicking enzyme amplification (NEAR), transcription-mediated amplification (TMA), and SMAP2 amplification. 18. The method of claim 14, wherein the target nucleic acid molecule is a methylated nucleic acid sequence, or the methylated nucleic acid sequence is a nucleic acid sequence obtained after converting unmethylated C to uracil. 19. Use of the Cas12b protein to prepare a kit for the detection of target nucleic acid molecules based on the bypass cleavage activity of single-stranded DNA. 20. Use of the Cas12b protein to prepare a reagent for the detection of target nucleic acid molecules based on the bypass cleavage activity of single-stranded DNA. 21. Application of the Cas12b protein in a one-step reaction system for detecting target nucleic acid molecules based on the bypass cleavage activity of single-stranded DNA, wherein the one-step reaction system includes a nucleic acid amplification reaction and a bypass cleavage reaction, wherein the nucleic acid amplification reaction and the bypass cleavage reaction are performed simultaneously . 22. A device for detecting the presence of target nucleic acid molecules in a sample, which includes: (1) an amplification reaction-bypass reaction module, which is used to perform nucleic acid amplification reactions and bypass cleavage reactions in a digital reaction system in which the bypass reaction is mediated by the Cas12b protein; And (2) a signal detection module that is used to detect whether a bypass cleavage reaction mediated by the Cas12b protein occurs in each digital reaction system, and the signal detection module includes an imaging module; wherein the nucleic acid amplification reaction and the bypass cleavage reaction are performed simultaneously; amplification reaction module - bypass cleavage reactions contains a temperature control unit to set the temperature of the digital reaction system in the device to a predetermined temperature; and the set temperature is 50-70°C, preferably 50-65°C, more preferably 55-65°C; the digital reaction system comprises a digital droplet PCR reaction system (ddPCR) or a digital PCR on chip reaction system (cdPCR); wherein the digital reaction system comprises a plurality of independent reaction system devices, and each reaction system device contains: (a) Cas12b protein; (b) a guide RNA, the guide RNA directing the Cas12b protein to specifically bind to target nucleic acid molecules, the target nucleic acid molecule being the target DNA; (c) a nucleic acid probe that is single stranded DNA; (d) buffer; (e1) a polymerase that is used to amplify the target DNA; (e2) an optional reverse transcriptase which is used for reverse transcription; (e3) dNTP, which is used for the amplification reaction and/or the reverse transcription reaction; And (f) sample for testing; however, in connection with the dilution processing, each independent reaction system contains either 1 copy of the nucleic acid from the sample to be tested, or 0 copies of the nucleic acid from the sample to be tested; And (3) sample loading module; And (4) control module. 23. The apparatus of claim 22, wherein the imaging module comprises a fluorescent detection device. 24. The apparatus of claim 22, wherein the control module performs an arithmetic analysis based on the number of fluorescent signals and the total number of reaction system devices, thereby obtaining a target nucleic acid quantification result.