CN115820818A - One-step nucleic acid detection method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a one-step nucleic acid detection method and application thereof. In the invention, a reaction system for DNA isothermal amplification RAA and a gene editing CRISPR/Cas12b fluorescent signal detection system are added simultaneously in the same space, and the step of transferring amplification products is not needed, so that the nucleic acid detection can be realized, the actual operation is simple and rapid, and the aerosol pollution is avoided. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, has the sensitivity of 0.2 copies/mu L, has extremely high sensitivity, can be used for detecting nucleic acid of bordetella pertussis, and is beneficial to reducing the infection rate and the disease transmission rate.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a one-step nucleic acid detection method and application thereof.
Background
Viral or bacterial respiratory tract infections are one of the most common diseases in humans worldwide, and identification of the causative agent of respiratory tract viral infections is of great importance for selection of appropriate therapeutic approaches and prevention of epidemics. Therefore, the development of a reagent for quickly and accurately diagnosing pathogens has a deep influence on disease monitoring, drug guidance and treatment evaluation.
Pertussis, an acute respiratory infection caused by bordetella pertussis, is an early diagnosis critical to the treatment of pertussis. Currently, the common laboratory methods for pertussis diagnosis include culture, direct fluorescent antibody detection, PCR, pairing, and single serological techniques (a.m. Wendelboe and a. Van Rie, expert review of molecular diagnostics, 2006, 6, 857-864). Isolation of bordetella pertussis by culture is the traditional "gold standard" for the diagnosis of pertussis. Its specificity is about 100%. However, the sensitivity of the culture is low, varying from 12% to 60%, and the culture takes a long time, on average, from 5 days to 10 days. The traditional Polymerase Chain Reaction (PCR) or reverse transcription polymerase chain reaction (RT-PCR) method is a gold standard for nucleic acid detection and has high sensitivity. Since the 90 s of the 20 th century, PCR has been used as a diagnostic tool for clinical bordetella pertussis and is widely used in research settings. However, the PCR technique requires skilled operators and a complete equipment infrastructure, and the sample detection time is long, which is not suitable for simple and rapid point-of-care testing (POCT).
In recent years, nucleic acid detection methods based on regular clustered short palindromic repeats/CRISPR associated proteins (CRISPR/Cas) have become hot spots for point-of-care detection (POCT). When RNA-guided CRISPR/Cas system proteins recognize a target nucleic acid, they can not only cleave in cis (cis-cleavage) the target nucleic acid, but also non-specifically cleave in trans (trans-cleavage) the flanking single-stranded non-target nucleic acid. By utilizing the characteristic, the CRISPR/Cas nuclease protein is combined with isothermal amplification technologies, such as Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP), researchers develop a series of sensitive, rapid and visual nucleic acid detection methods, and the methods do not need special precise instruments. For example, SHERLOCK based on the specificity of Cas13a or Cas13b with high sensitivity; cas12 a-based dnazymes target the DETECTR of CRISPR and Cas12 b-based Cas12 b-mediated DNA detection cdelection. However, these methods require a two-step process, first amplifying the target nucleic acid sequence, followed by CRISPR detection. Such multiple processing steps increase operating time, cross-contamination and the risk of aerosol generation.
Disclosure of Invention
Aiming at the technical problems, the invention provides a one-step nucleic acid detection method, which can simultaneously add two reaction systems in the same space without an additional amplification product transfer step, has simple and rapid actual operation, avoids aerosol pollution and avoids cross pollution.
In order to achieve the technical effects, the invention adopts the technical scheme that:
a one-step method for detecting nucleic acid, comprising the steps of:
s1, mixing and oscillating a target detection sample and a releasing agent to obtain a mixture I;
s2, simultaneously adding a DNA constant-temperature amplification system RAA containing a specific primer designed aiming at a target sequence and a gene editing CRISPR/Cas12b system containing a specific sgRNA designed aiming at the target sequence and a fluorescent report probe into a PCR reaction tube;
s3, adding a detection sample and a mixture I of the detection sample and a releasing agent into the reaction system, performing vortex oscillation, and performing short-time centrifugation (instantaneous centrifugation by using a palm centrifuge) to obtain a mixture II;
and S4, placing the mixture II obtained in the step S3 into a PCR instrument for reaction, synchronously reading fluorescence, and judging results according to fluorescence signals to obtain the fluorescent material.
Preferably, the PCR reaction tube in step S2 may also be a constant temperature heater for reading fluorescence signals, a constant temperature nucleic acid amplification instrument, or an end point fluorescence interpretation under LED blue light or ultraviolet light after being put into the constant temperature heater.
The equipment matched with the reaction system is not limited to a PCR instrument, but also comprises a constant temperature heater or a constant temperature nucleic acid amplification instrument for reading a fluorescent signal, or is put into the constant temperature heater at 37 ℃ for 30min and then subjected to end point fluorescent interpretation under an LED blue light or ultraviolet lamp.
Preferably, the DNA isothermal amplification system RAA in step S1 further includes a molecular crowding reagent, a nucleic acid amplification promoter, a reaction dry powder enzyme preparation, and an amplification primer.
Preferably, the molecular crowding reagent is 20000 polyethylene glycol (including but not limited to); the nucleic acid amplification initiator is 280mM magnesium acetate (including but not limited to).
Preferably, the gene editing CRISPR/Cas12b system of step S2 further comprises a Cas12b enzyme, sgrnas, and a fluorescent probe.
Preferably, the fluorescent probe is provided with a fluorescent group and a quenching group.
Preferably the fluorophore is FAM (including but not limited to); the quencher group is BHQ1 (including but not limited to); the fluorescent probe is FAM-CCCCCC-BHQ1.
Preferably, the reaction process in the step S4 is carried out at 30-42 ℃ for 25-35min.
The invention also provides application of the detection method in preparation of a Bordetella pertussis detection kit.
The invention also provides a bordetella pertussis detection kit, which comprises a DNA constant temperature amplification RAA system, a gene editing CRISPR/Cas12b fluorescent signal detection system and an instruction for use.
In the invention, a one-step nucleic acid detection method Rcod is provided, the RAA and the CRISPR/Cas12b fluorescent signal detection for gene editing are simultaneously carried out in one step, an additional amplification product transfer step is not needed, the actual operation is simple and rapid, and the aerosol pollution is avoided. Furthermore, the one-step nucleic acid detection method Rcod provided by the invention has universality, and can be used for detecting cells, viruses, bacteria and the like containing target nucleic acid.
The inventors firstly and separately explore trans-cleavage activity and cis-cleavage activity of CRISPR/Cas12b in a very wide temperature range of 25 ℃ to 72 ℃. It is confirmed that the CRISPR/Cas12b shows extremely strong trans-cleavage activity in the temperature range of 30-68 ℃, and the CRISPR/Cas12b has extremely strong cis-cleavage activity in the temperature range of 45-60 ℃. Further combining the optimal reaction temperature range of 30-42 ℃ for DNA isothermal amplification RAA, finally determining that the optimal reaction temperature range of one-step nucleic acid detection is 30-42 ℃, and the alicyclic acidophilus Cas12b (AaacCas 12 b) still has high trans-cleavage activity in the temperature range, so that a specific cleavage probe can be obtained, and the whole reaction system has extremely high specificity; the cis-form cutting activity is very weak, so that DNA constant-temperature amplification products cannot be cut too much, and the sensitivity of a one-step nucleic acid detection system is ensured. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, and the sensitivity reaches 0.2 copies/mu L. 221 bordetella pertussis clinical samples were detected, and the sensitivity and specificity of the invention were 97.96% and 99.19% respectively, comparing the PCR detection results.
The one-step nucleic acid detection method Rcod provided by the invention does not need an additional step of transferring an amplification product, is simple and rapid in actual operation, and avoids aerosol pollution.
The primer pair, sgRNA and the probe used in the invention are obtained by screening a large number of primers, and the detection specificity and the sensitivity of the primer pair are high, and the detection lower limit IS 0.2 DNA copies per microliter. The invention adopts RAA-CRISPR/Cas12b technology to establish a one-step method for rapidly detecting bordetella pertussis, has high sensitivity and high specificity of molecular biological detection, is simple and convenient to operate, does not need to depend on complex instruments, and completely meets the requirement of bedside detection of bordetella pertussis.
The technical scheme of the invention can obviously improve the detection speed. Compared with the conventional PCR, the three steps of denaturation, annealing and extension are not needed, the RAA-CRISPR/Cas12b is synchronously reacted at 37 ℃, and the reaction can be completed within 30min. The detection method does not need complex instruments and equipment, is not easy to cause aerosol pollution, and is suitable for field detection. The detection method can amplify and perform visual end point fluorescence detection under the conditions of normal temperature and constant temperature, does not need to depend on complex instruments and equipment such as a PCR instrument, a fluorescence quantitative PCR instrument, an electrophoresis tank and the like, does not need complex sample treatment, can release sample nucleic acid by a release agent, can detect the nucleic acid by anyone according to the instruction, and can really realize portable field rapid nucleic acid detection.
The kit for rapidly detecting bordetella pertussis is established by adopting the one-step method RAA-CRISPR/Cas12b technology for the first time, can be used for clinical field detection through specificity, sensitivity and actual sample analysis, and provides a sensitive and reliable new method for the instant detection of bordetella pertussis.
Compared with the prior art, the nucleic acid detection method provided by the invention has the advantage that two reaction systems are simultaneously carried out in one step. The method comprises a reaction system I used for DNA isothermal amplification RAA and a reaction system II used for gene editing CRISPR/Cas12b fluorescent signal detection. The nucleic acid detection method provided by the invention can be used for simultaneously adding two reaction systems in the same space without an additional step of transferring amplification products, is simple and quick in actual operation, and avoids aerosol pollution. The nucleic acid detection method provided by the invention can reduce the whole process from sample to nucleic acid result interpretation to within 30min, and the system does not need to extract nucleic acid, has the sensitivity of 0.2 copies/mu L and has extremely high sensitivity. The nucleic acid detection method provided by the invention has the characteristics of simple operation, no aerosol pollution, no toxic substance, short reaction time, high reaction sensitivity and the like, can greatly meet the requirements of Bordetella pertussis bedside detection POCT, is beneficial to early diagnosis and early treatment, and reduces the infection rate and the disease transmission rate.
Drawings
FIG. 1 is a schematic diagram of a nucleic acid detection method according to an embodiment of the present invention;
FIG. 2 shows the results of fluorescence signals of Rcod obtained by the one-step nucleic acid detection method of the present invention under different temperature conditions;
FIG. 3 is a diagram showing the result of the lowest detection limit for detecting B.pertussis by the one-step nucleic acid detection method Rcod according to the present invention;
FIG. 4 is a graph showing the results of detecting pertussis clinical specimens by the one-step nucleic acid detection method Rcod of the present invention.
Detailed Description
The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Example 1 one-step method for nucleic acid detection
As shown in figure 1, the one-step nucleic acid detection method Rcod comprises the steps of using the Rcod in DNA isothermal amplification RAA and gene editing CRISPR/Cas12b fluorescent signal detection. In the same reaction system, selecting target sequences according to different target nucleic acid molecules to be detected in each target detection sample, designing corresponding specific primers aiming at the target sequences of the target nucleic acid molecules to be detected in each target detection sample, and taking the target detection sample added with a release agent as a target nucleic acid template; meanwhile, a target sequence is selected according to different target nucleic acid molecules to be detected in each target detection sample, corresponding sgRNA is designed aiming at the target sequence, and a single-stranded base sequence with a fluorescent group and a quenching group is used as a fluorescent report probe.
1.1. Design of primers and sgRNA
The IS1663 gene (with the sequence ID being CP 046995.1) of Bordetella pertussis (the gene sequence can be used as a conserved sequence) IS adopted as a target sequence, and specific primers for amplifying the target sequence are as follows:
an upstream primer FP: CCTACGGGTCTGTATCACGAGCAAGCGGC;
a downstream primer RP: GCGTCTGTCCATAGCGAGCCAGCACGTAGC;
the specific sgRNA (AacCas 12 b) is:
GGUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACCCAAGGCCUUGGCGCUGCGC；
non-specific fluorescent probe P: FAM-CCCCCC-BHQ1.
1.2. Extraction of pathogen DNA
The nasopharyngeal swab is firstly put into 1mL of 0.9% normal saline for elution, and then the eluent is mixed with the release agent with the same volume.
Extracting pathogen DNA without a special nucleic acid extraction kit, instantly centrifuging 20 μ L of a target detection sample and a release agent (with main components of NaOH, tris-acetic acid pH8.0, dextran, mannitol, sucrose, etc., and the addition amount of 20 μ L) on a palm centrifuge, mixing and shaking;
the release agent is added according to the following formula materials: 5mL of 2M NaOH; 5mL of 1M Tris-acetate (pH8.0); 2.5mL of glucan with the mass concentration of 20%; 5mL of mannitol with the mass concentration of 12.5%; 2.5mL of sucrose with the mass concentration of 20 percent; adding water to make up to 50mL, mixing uniformly and storing at normal temperature for later use.
3. Configuration of one-step nucleic acid detection system
The upstream primer FP (10. Mu.M, added at 0.8. Mu.L), the downstream primer RP (10. Mu.M, added at 0.8. Mu.L), the specific sgRNA (AacCas 12b, 0.3. Mu.g/. Mu.L, 1. Mu.L), the non-specific fluorescent probe P (25mM, 1. Mu.L) in step 2 were added simultaneously to the PCR reaction tube, while the molecular crowding reagent 6.25. Mu.L (which may be but is not limited to 20, 000 polyethylene glycol (PEG)), the nucleic acid amplification initiator 1.25. Mu.L (which may be but is not limited to 280mM magnesium acetate), the reaction dry powder enzyme preparation (purchased from Hangzhou Biotech, RAA Freeze-drying reagent enzyme, one system added with half tube) and Cas12b enzyme (2 mg/mL, 1. Mu.L), and water was added to 20. Mu.L.
Then taking 5 mu L of a detection sample mixed with the releasing agent, adding the detection sample into the reaction system, and carrying out vortex oscillation and short-time centrifugation;
and (3) putting the mixed one-step reaction system into a PCR instrument, setting the temperature to be 37 ℃, and reacting for 30min.
4. Results of the experiment
Real-time fluorescence reading can be carried out within 30min of the reaction, or end point fluorescence interpretation is carried out under LED blue light or an ultraviolet lamp after 30min, and result interpretation is carried out according to fluorescence signals.
Example 2 optimal reaction temperature selection
Since AacCas12b in example 1 shows weak cis-cleavage activity at a lower temperature, this example tested the optimal reaction temperature of the one-step nucleic acid detection method Rcod established in example 1 at a temperature of 30 ℃ to 45 ℃. The pUC57-IS1663 plasmid containing the Bordetella pertussis IS1663 sequence was diluted to a concentration of 200 copies/. Mu.L with TE buffer and used as a target sequence.
As shown in FIG. 2, the whole system was incubated at 37 ℃ for 10min and then the temperature was raised to 48 ℃ for 20min (37-48 ℃); or reacting at 30 deg.C, 32 deg.C, 35 deg.C, 37 deg.C, 39 deg.C, 42 deg.C and 45 deg.C for 30min.
The results are shown in FIG. 2, in which FIG. 2a shows the real-time fluorescence signal monitored within 30min of the reaction, and FIG. 2b shows the end-point fluorescence detection performed after 30min of the reaction. As can be seen from FIG. 2, the one-step nucleic acid detection method Rcod showed a significantly enhanced fluorescence signal at a temperature of 32 ℃ to 39 ℃. In addition, the fluorescent signal of the constant temperature reaction at 37 ℃ for 30min is almost consistent with that of a control group (firstly, the reaction is carried out at 37 ℃ for 10min for amplification, and then the temperature is increased to 48 ℃ for CRISPR fluorescence detection for 20 min). After incubation for 30min, the end-point fluorescence signals showed consistent results by LED blue or uv irradiation. Therefore, we chose 37 ℃ as the optimal reaction temperature for the one-step nucleic acid detection system.
Example 3 lowest detection limit LOD test
This example performed the lowest limit of detection LOD test for the one-step nucleic acid detection method for amplification and detection of bordetella pertussis rdson established in example 1. The method comprises the following steps:
4 replicates of each gradient were performed using a series of 10-fold dilutions of the Bordetella pertussis IS-1663 plasmid (i.e., pUC57-IS1663 in example 2) as template. The samples were also diluted in TE buffer and the concentration of the target sequence template was 2X 10 3 、2×10 2 、2×10 1 、2×10 0 、2×10 -1 And negative control NTC. Respectively monitoring real-time fluorescence signals within 30min of reaction (figure 3 a), carrying out end-point fluorescence detection after 30min of reaction (figure 3 b), and then carrying out end-point fluorescence shooting through LED blue light irradiation or ultraviolet irradiation after 30min of reaction.
As can be seen from FIG. 3, the real-time fluorescence and the LED blue/ultraviolet end-point fluorescence result are consistent, showing that the one-step nucleic acid detection system can stably detect input plasmid DNA as low as 0.2 copy/. Mu.L at 37 ℃, and has extremely high sensitivity.
Example 4 clinical testing
This example performed a true clinical sample test on the one-step nucleic acid detection method for amplification and detection of bordetella pertussis rdo established in example 1. The method comprises the following steps:
to verify the clinical applicability of the Rcod platform, 98 pertussis-cultured positive samples and 123 pertussis-cultured negative samples of beijing child hospital affiliated to the university of capital medicine were tested by a one-step nucleic acid detection method Rcod and a real-time fluorescent quantitative PCR method, respectively. The infection status of the patients was independently confirmed by the Beijing Children hospital affiliated with the university of capital medical science. The 221 samples were divided into two parts, one part for the one-step nucleic acid detection method Rcod and the other part for real-time fluorescent quantitative PCR (these 2 methods all detected 221 samples). In addition, in order to facilitate the rapid detection of clinical samples, the sample is processed by using a nucleic acid releasing agent method (namely, the sample to be tested is mixed with the releasing agent), and the sample extraction is completed within 5 seconds. Meanwhile, detecting by using a bordetella pertussis nucleic acid detection kit (PCR fluorescent probe method); the kit can be purchased from Shenzhen hundred million cubic biotechnology Limited company, the production license number is Yuejie Yaojian production license 20142667, the national mechanical registration number is 20203400152, the latest approval date of the specification is 2020, 2 and 14 days, and the real-time PCR test process is operated according to the latest approval specification.
As shown in fig. 4, wherein (a, b) a total of 221 pertussis clinical specimens, (a) 98 positive specimens, (b) 123 negative specimens; (c) Fluorescence values of pertussis-positive patients (+) and control groups (-). (d) Waterfall distribution of all bordetella pertussis clinical samples (n = 221). (e) Detecting bordetella pertussis in clinical samples, wherein the detection result is directly visualized under LED blue light and ultraviolet light.
FIGS. 4a-b show that we compared the raw fluorescence intensity of the one-step nucleic acid detection method Rcod with the cycle threshold (Ct) of real-time fluorescent quantitative PCR, which is proportional to the concentration of the target gene. Both detection methods showed good agreement. 97 pertussis positive culture samples were identified as positive for B.pertussis by our one-step nucleic acid detection method Rcod with a raw fluorescence intensity greater than 2699a.u. (3X average of initial fluorescence values for all samples) (FIGS. 4a, c-d). In addition, our one-step nucleic acid detection method Rcod identified 124 pertussis culture negative samples as negative for b.pertussis with an endpoint fluorescence value of less than 2000a.u. (fig. 4 b-d). In addition, the end point fluorescence detection under blue or ultraviolet illumination of the LED showed consistent results (fig. 4 e).
In addition, we calculated the sensitivity and specificity of the one-step nucleic acid detection method Rcod for detecting Bordetella pertussis clinical samples. Compared with real-time fluorescent quantitative PCR, our one-step nucleic acid detection method Rcod shows 97.96% sensitivity, 99.19% specificity and 98.64% accuracy, with a detection time of 30min, and real-time fluorescent quantitative PCR of 2 hours (Table 1). The one-step nucleic acid detection method Rcod has greater application value in clinical diagnosis and detection. Can be used for clinical field detection, and provides a sensitive and reliable new method for the instant detection of bordetella pertussis.
TABLE 1 comparison of the identity between the detection method of the invention and real-time PCR for 221 clinical samples
Finally, it should be noted that the above-mentioned embodiments are only illustrative for the principle, performance and efficacy of the present invention, and are not meant to limit the present invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A one-step nucleic acid detection method is characterized by comprising the following steps:
s1, mixing and oscillating a target detection sample and a releasing agent to obtain a mixture I;
s2, simultaneously adding a DNA constant-temperature amplification system RAA containing a specific primer designed aiming at a target sequence and a gene editing CRISPR/Cas12b system containing a specific sgRNA designed aiming at the target sequence and a fluorescent report probe into a PCR reaction tube;
s3, adding a mixture I of a detection sample and a release agent into the reaction system, performing vortex oscillation, and performing short-time centrifugation to obtain a mixture II;
and S4, placing the mixture II obtained in the step S3 into a reaction instrument for reaction, reading fluorescence, and judging results according to fluorescence signals to obtain the fluorescent material.
2. The detection method according to claim 1, wherein the reaction apparatus of step S4 is a PCR reaction tube, and has one of a constant temperature heater for reading a fluorescence signal and a constant temperature nucleic acid amplification apparatus; the manner of reading fluorescence is as follows: and (3) directly and synchronously reading fluorescence according to PCR reaction, or putting the PCR product into a constant temperature heater and then performing end point fluorescence interpretation under LED blue light or an ultraviolet lamp.
3. The detection method according to claim 1, wherein the DNA isothermal amplification system RAA of step S2 further comprises a molecular crowding reagent, a nucleic acid amplification primer, a reaction dry-powder enzyme preparation, and an amplification primer.
4. The detection method according to claim 3, wherein the molecular crowding reagent is polyethylene glycol having a molecular weight of 20000; the nucleic acid amplification initiator is 280mM magnesium acetate.
5. The detection method of claim 1, wherein the gene editing CRISPR/Cas12b system of step S2 further comprises a Cas12b enzyme, sgrnas and fluorescent probes.
6. The detection method of claim 5, wherein the fluorescent probe comprises a fluorophore and a quencher.
7. The detection method according to claim 6, wherein the fluorescent group is FAM; the quenching group is BHQ1; the fluorescent probe is FAM-CCCCCC-BHQ1.
8. The detection method according to claim 1, wherein the reaction process in the step S4 is carried out at 30-42 ℃ for 25-35min.
9. Use of the detection method according to any one of claims 1 to 8 in the preparation of a bordetella pertussis detection kit.
10. A Bordetella pertussis detection kit is characterized by comprising a DNA constant temperature amplification RAA system and a gene editing CRISPR/Cas12b fluorescent signal detection system, and the kit adopts the detection method of any one of claims 1-8 to detect a sample to be detected.
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