CA3059288A1

CA3059288A1 - Novel colorimetric sensing method for dual pathogen

Info

Publication number: CA3059288A1
Application number: CA3059288A
Authority: CA
Inventors: Yunbo LUO; Wentao Xu; Kunlun HUANG; Jingjing TIAN; Zaihui DU
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-02-08
Filing date: 2018-08-08
Publication date: 2019-08-15
Anticipated expiration: 2038-08-08
Also published as: WO2019153675A1; CN108251514A; CA3059288C

Abstract

Provided in the present application is a novel colorimetric sensing method for a dual pathogen, comprising an in vitro nucleic acid amplification and a detection method, where the in vitro nucleic acid amplification uses a primer consisting sequentially of complementary sequences, a connecting arm, complementary sequences, and a nucleic acid sequence of a specifically amplified target to be tested for a superfast PCR reaction, and the detection uses a hairpin sequence consisting of complementary sequences and a G-quadruplex to implement self-assembly color development.

Description

NOVEL COLORIMETRIC SENSING METHOD FOR DUAL PATHOGEN
This application claims the priority to Chinese Patent Application No.
201810128946.4 titled "NOVEL COLORIMETRIC SENSING METHOD FOR DUAL PATHOGEN", filed with the Chinese State Intellectual Property Office on February 08, 2018, which is incorporated herein by reference in its entirety.
FIELD
[0001] The present disclosure relates to the technical field of biological detection, specifically to a novel colorimetric sensing method for dual pathogen.
BACKGROUND

[0002] The traditional bacterial detection method is mainly based on the physiological and biochemical characteristics of bacteria. After the steps of pre-enrichment, selective plate separation and biochemical identification, it takes 5-7 days from sampling to determination. The detection period is long, the operation is cumbersome and the workload is large. It has been more than half a century since the specificity of the antigen-antibody reaction has been used to identify bacteria, but the screening of microbial antibodies is cumbersome and the specificity of the final detection is not high. The continuous improvement and development of molecular biology detection technology has overcome the problems of tedious and time-consuming experimentation of traditional detection methods and led to the rapid development of rapid detection methods for microorganisms, but the disadvantage of molecular biology methods is that the results are not visualized, making the results analysis not easy.
SUMMARY

[0003] The novel colorimetric sensing method established by the present disclosure overcomes the deficiencies of the existing detection technologies and realizes accurate, rapid, simple and - -13718731.2 efficient detection and analysis of microorganisms.

[0004] The present disclosure aims to provide a detection method, comprising nucleic acid amplification in vitro, wherein the reaction system of the nucleic acid amplification in vitro comprises a upstream primer and a downstream primer, wherein the upstream primer comprises a complementary sequence A, a linking arm, a complementary sequence B, and a nucleotide sequence capable of specifically amplifying a target to be detected;
the linking arm is provided between the complementary sequence A and complementary sequence B, and the nucleotide sequence capable of specifically amplifying the target to be detected is provided at 5' terminal or 3' terminal of the upstream primer;
the nucleotide sequence of the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or reverse complementary to each other;
the linking arm comprises a moeity capable of inhibiting polymerase binding and/or a moeity capable of inhibiting new strand extension during in vitro nucleic acid amplification; and the downstream primer comprises the nucleotide sequence capable of specifically amplifying the target to be detected.

[0005] The A and B are only used to distinguish different complementary sequences, and are not used for sorting.

[0006] The complementation includes complementation or reverse complementation as defined by the conventional art or common general knowledge, and/or performing complementation or reverse complementation according to complementary principles as defined by the conventional art or common general knowledge.

[0007] The polymerase includes a polymerase that can be used for the in vitro nucleic acid amplification.

13718731.2

[0008] The nucleotide sequence capable of specifically amplifying the target to be detected comprises a primer sequence designed according to a characteristic sequence of the target to be detected; the characteristic sequence comprises the characteristic sequence defined by the conventional art or common general knowledge; and the design comprises the design method recorded by the conventional art or common general knowledge.

[0009] Specifically, the method further comprising at least one of the following 1) to 2):
1) the nucleic acid amplification in vitro includes ultra-rapid PCR, wherein the reaction process of the ultra-rapid PCR comprises: 90-98 C for 2-6s and 50-60 C for 2-8s, for 20-40 cycles in total;
specifically, the reaction process of the ultra-rapid PCR reaction comprises:
95 C for 4s;
58 C for 6s; and a total of 30 cycles;
specifically, the concentration of the upstream primer and the downstream primer in the reaction system of the ultra-rapid PCR reaction is more than 10 times of the concentration of the regular PCR; specifically, the concentration is 20 times; the reaction system of the ultra-rapid PCR reaction further comprises a DNA polymerase, and the concentration of the DNA
polymerase is more than 10 times of the concentration of the regular PCR, more specifically, the concentration is 60 times; and 2) the linking arm comprises a compound having a long-chain structure.

[0010] More specifically, the linking arm is oxyethyleneglycol, and the chemical structure of oxyethyleneglycol is:
0-P _____________________________________________________________ 0 (I)

[0011] Specifically, the method further comprising at least one of the following 1) to 8):
1) the upstream primer includes a primer obtained by linking the nucleotide sequences set 13718731.2 forth in SEQ ID NO: 1 and SEQ ID NO: 2 via the linking arm;
2) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 3;
3) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: 1 and/or SEQ ID NO: 2 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 1 and/or SEQ ID
NO: 2;
4) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 3 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
3;
5) the upstream primer includes a primer obtained by linking the nucleotide sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5 via the linking arm;
6) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 6;
7) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: 4 and/or SEQ ID NO: 5 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 4 and/or SEQ ID
NO: 5; and 8) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 6 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
6.

[0012] The "function" refers to realizing specific amplification or detecting the target to be detected.

[0013] More specifically, the upstream primer is:
AGA GAGAGAGAGGGAAAGAGAGAG- ox yethyleneglycol-CTCTCTCTTTCCCTCTCT
CTCTCTTTITTGTGAAATTATCGCCACGTTCGGGCAA and/or TGAGGTAGTAGGTTGTATAGTT-oxyethylenegl ycol-AACTATACAAC CTACTACCTCA

13718731.2 AAAAAAAAAAAGCACATAACAAGCG.

[0014] The upstream primers can be synthesized commercially, and the preparation method thereof belongs to conventional art.

[0015] Another object of the present disclosure is to provide a detection method, comprising a color reaction based on nucleic acid self-assembly, wherein the reaction system of the color reaction based on nucleic acid self-assembly comprises a hairpin sequence which comprises all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence, a complementary sequence C, and a complementary sequence D, wherein the all or a part of the nucleotide sequence containing a G-quadruplex forming nucleotide sequence is provided at 5' terminal and/or 3' terminal of the hairpin sequence;
the complementary sequence C and the complementary sequence C of the other hairpin sequence are complementary and/or reverse complementary to each other; and the complementary sequence D and the target to be detected or the nucleotide sequence linked to the target to be detected are complementary and/or reverse complementary.

[0016] The C and D are only used to distinguish different complementary sequences, and are not used for sorting.

[0017] The G-quadruplex forming nucleotide sequence comprises the G-quadruplex forming nucleotide sequence as defined by the conventional art or common general knowledge.
Specifically, the G-quadruplex forming nucleotide sequence comprises the sequence capable of self-assembling to form a G-quadruplex structure having an activity similar to horseradish peroxidase, and the G-quadruplex structure, under the induction of hemin, catalyzes ABTS2- and H202 to form ABTS- , which makes the reaction solution blue-green.

[0018] Specifically, the hairpin sequence can cleave the G-quadruplex forming nucleotide sequence at a ratio of 25% and/or 75%, and the cleaved sequences are respectively added at the 5' terminal and/or the 3' terminal of the hairpin sequence; more specifically, a T base is further added after the cleaving; the T base is added at the 5' terminal and/or the 3' terminal of the cleaved sequence.

[0019] Specifically, the G-quadruplex forming nucleotide sequence includes the nucleotide 13718731.2 sequences set forth in SEQ ID NO: 7 and/or SEQ ID NO: 8.

[0020] Specifically, the complementary sequence D may be complementary and/or reverse complementary to the complementary sequence A and/or complementary sequence B
according to the present disclosure.

[0021] More specifically, the hairpin sequence comprises at least one of the following 1) to 4):
1) the nucleotide sequence set forth in SEQ ID NO: 9 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ II) NO: 9 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 9;
2) the nucleotide sequence set forth in SEQ ID NO: 10 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 10 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 10;
3) the nucleotide sequence set forth in SEQ ID NO: 11 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 11 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 11; and 4) the nucleotide sequence set forth in SEQ ID NO: 12 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 12 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 12.

[0022] More specifically, the color reaction based on nucleic acid self-assembly further comprises at least one of the following 1) to 2):
1) the reaction condition of the color reaction based on nucleic acid self-assembly comprises a step of incubating at 37 C for 20 mm; and 2) the final concentration of the hairpin sequence in the reaction system of the color reaction based on nucleic acid self-assembly is 2 M.

[0023] Another object of the present disclosure is to provide a detection method, comprising first amplifying the target to be detected by any of the methods according to the present 13718731.2 disclosure, and then detecting the target to be detected by any of the methods according to present disclosure.

[0024] Specifically, in the method, the complementary sequence D according to the present disclosure and the complementary sequence A and/or complementary sequence B
according to the present disclosure are complementary or reverse complementary.

[0025] Specifically, the method further comprising at least one of the following 1) to 4):
1) determining whether an object to be detected contains the target to be detected by a color change of final reaction system;
specifically, when the color of the reaction system changes, the object to be detected is determined to contain the target to be detected; more specifically, when the color of the reaction system turns blue-green, the object to be detected is determined to contain the target to be detected;
2) calculating the concentration of the target to be detected in the object by a method of plotting standard curve according to the color of the final reaction system;
3) amplifying the detection signal of the target to be detected by increasing the type of hairpin sequence in the reaction system;
specifically, for each additional hairpin sequence, the signal is amplified 2-4 times;
4) achieving dual or multiple detection by increasing the type of upstream primer or downstream primer, and simultaneously increasing the type of hairpin sequence in the reaction system.

[0026] Specifically, when the detection is for dual or multiple detection, the microarray method can be used to determine whether the object to be detected contains the target to be detected or contains several targets to be detected. The microarray method comprises separately placing the different types of hairpin sequences in different wells to perform reactions, and then determining whether the sample contains the target to be detected or several targets to be detected according to the reaction results. When the color of the reaction liquid changes or turns blue-green, it is determined that the target to be detected is present; the total number of wells in which the color 13718731.2 change or the blue-green color is generated is the total number of targets which are contained in the sample to be detect.

[0027] The types of hairpin sequence: if the complementary sequence D is complementary or reverse complementary to the complementary sequence A and/or B in the same upstream primer, then they are the same type of hairpin sequence. Otherwise, the sequence is different type of hairpin sequence; and the upstream primers having identical nucleotide sequences are the same upstream primers.

[0028] The types of the upstream primer or the downstream primer: a primer pair that can amplify the same target to be detected are the same type of upstream primer or downstream primer; otherwise, the primer pair that can amplify different targets to be detected are different types of upstream primer or downstream primer.

[0029] Another object of the present disclosure is to provide a kit and/or a biosensor, comprises at least one of the following 1) to 2):
1) the upstream primer and the downstream primer, the upstream primer comprises a complementary sequence A, a linking arm, a complementary sequence B, and a nucleotide sequence capable of specifically amplifying a target to be detected, wherein the linking arm is provided between the complementary sequence A and complementary sequence B, and the nucleotide sequence capable of specifically amplifying the target to be detected is provided at 5' terminal or 3' terminal of the upstream primer;
the nucleotide sequence of the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or reverse complementary to each other;
the linking arm comprises a moeity capable of inhibiting polymerase binding and/or a moeity capable of inhibiting new strand extension during in vitro nucleic acid amplification; and the downstream primer comprises the nucleotide sequence capable of specifically amplifying the target to be detected; and 2) a hairpin sequence comprising all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence, a complementary sequence C, and a complementary sequence D, wherein the all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence is provided at 5' terminal and/or 3' terminal of the hairpin sequence;
the complementary sequence C and the complementary sequence C of the other hairpin sequence are complementary and/or reverse complementary; and the complementary sequence D and the target to be detected or the nucleotide sequence linked to the target to be detected are complementary and/or reverse complementary.

[0030] The complementation includes complementation or reverse complementation as defined by the conventional art or common general knowledge, and/or performing complementation or reverse complementation according to complementary principles as defined by the conventional art or common general knowledge.

[0031] The polymerase includes a polymerase that can be used for the in vitro nucleic acid amplification.

[0032] The nucleotide sequence capable of specifically amplifying the target to be detected specifically comprises a primer sequence designed according to a characteristic sequence of the target to be detected; the characteristic sequence comprises the characteristic sequence defined by the conventional art or common general knowledge; and the design comprises the design method recorded by the conventional art or common general knowledge.

[0033] Specifically, the linking arm comprises a compound having a long-chain structure.

[0034] More specifically, the linking arm is oxyethyleneglycol, and the chemical structure of oxyethyleneglycol is:

13718731.2 C)

[0035] Specifically, the upstream primer and the downstream primer comprise at least one of the following 1) to 8):
1) the upstream primer includes a primer obtained by linking the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 via the linking arm;
2) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 3;
3) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: 1 and/or SEQ ID NO: 2 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 1 and/or SEQ ID
NO: 2;
4) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 3 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
3;
5) the upstream primer includes a primer obtained by linking the nucleotide sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5 via the linking arm;
6) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 6;
7) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: 4 and/or SEQ ID NO: 5 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 4 and/or SEQ ID
NO: 5; and 8) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 6 by substituting, adding and/or deleting one or 13718731.2 more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
6.

[0036] The "function" refers to realizing specific amplification or detecting the target to be detected.

[0037] More specifically, the upstream primer is:
AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol-CTCTCTCTTTCCCTCTCT
CTCTCTTTTTTGTGAAATTATCGCCACGTTCGGGCAA
and/or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol-AACTATACAACC
TACTACCTCAAAAAAAAAAAAGCACATAACAAGCG.

[0038] The C and D are only used to distinguish different complementary sequences, and are not used for sorting.

[0039] The G-quadruplex forming nucleotide sequence comprises the G-quadruplex forming nucleotide sequence as defined by the conventional art or common general knowledge.
Specifically, the G-quadruplex forming nucleotide sequence comprises the sequence capable of self-assembling to form a G-quadruplex structure having an activity similar to horseradish peroxidase, and the G-quadruplex structure, under the induction of hemin, catalyzes ABTS2 and H202 to form ABTS- , which makes the reaction solution blue-green.

[0040] Specifically, the hairpin sequence can cleave the G-quadruplex forming nucleotide sequence at a ratio of 25% and/or 75%, and the cleaved sequences are respectively added at the 5' terminal and/or the 3' terminal of the hairpin sequence; more specifically, a T base is further added after the cleaving; the T base is added at the 5' terminal and/or the 3' terminal of the cleaved sequence.

[0041] Specifically, the G-quadruplex forming nucleotide sequence includes the nucleotide sequences set forth in SEQ ID NO: 7 and/or SEQ ID NO: 8.

[0042] Specifically, the complementary sequence D is complementary or reverse complementary to the complementary sequence A and/or B.

[0043] More specifically, the hairpin sequence comprises at least one of the following 1) to 4):
1) the nucleotide sequence set forth in SEQ ID NO: 9 and/or a nucleotide sequence which is 13718731.2 modified from the nucleotide sequence set forth in SEQ ID NO: 9 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 9;
2) the nucleotide sequence set forth in SEQ ID NO: 10 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 10 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 10;
3) the nucleotide sequence set forth in SEQ ID NO: 11 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 11 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 11; and 4) the nucleotide sequence set forth in SEQ ID NO: 12 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 12 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ NO: 12.

[0044] Specifically, the kit and/or the biosensor comprises the following 1) to 3):
1) AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge - CTCTCT
CTTTCCCTCTCTCTCTCTTTTTTGTGAAATTATCGCCACGTTCGGGCAA
and/or TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol bridge - AACTATACAACCTACTA
CCTCAAAAAAAAAAAAGCACATAACA;
2) TCATCGCACCGTCAAAGGAACC and/or GATAAAGAAGAAACCAGCAG; and 3) at least one of the nucleotide sequences set forth in SEQ ID NO:9, SEQ ID
NO:10, SEQ
ID NO:11, and SEQ ID NO:12;
wherein, the chemical structure of oxyethyleneglycol is:

13718731.2 0=-P ____________________________________________________________ 0

[0045] Another object of the present disclosure is to provide the use of any one of the methods according to the present disclosure, and any one of the kits and/or the biosensors according to the present disclosure.
.. [0046] Specifically, the use comprises at least one of the following 1) to 4):
1) detecting microorganisms;
2) preparing products for use in microorganism detection and/or related products;
3) detection of dual or multiple microorganisms; and 4) preparing products for use in dual or multiple microbial detection and/or related products.
[0047] Specifically, the microorganisms include Salmonella and/or Staphylococcus aureus.
[0048] Optionally, the use does not include the diagnosis and treatment of the disease described in Article 25 of the Chinese Patent Law.
[0049] A novel dual colorimetric sensing method based on ultra-rapid PCR is established by the present disclosure:
(1) this method establishes an ultra-rapid PCR reaction system that reduces the traditional PCR process, which takes about 3 hours, to 5 minutes, significantly reducing the time spent on PCR reactions;
(2) the ultra-rapid PCR reaction system is equipped with an enzyme-linked color module based on nucleic acid self-assembly, which not only amplifies the reaction signal again to .. facilitate the ultra-sensitive detection of pathogenic bacteria but also solves the problem that the traditional PCR results are difficult to visually detect; and (3) color reaction system is set in wells and sorted by microarray to solve the problem of 13718731.2 dual detection for pathogenic bacteria.
[0050] In one embodiment of the present disclosure, amplification primers for ultra-rapid polymerase chain reaction (PCR) are designed based on virulence genes of Salmonella and Staphylococcus aureus, and a color module based on nucleic acid self-assembly is combined to integrate and establish a novel dual colorimetric sensing method based on ultra-rapid PCR for ultrasensitive detection of Salmonella and Staphylococcus aureus.
[0051] The present disclosure has the following beneficial technical effects:
1) the detection method and the biosensor established by the present disclosure are faster and more sensitive than the conventional method, and have the advantages of high specificity, high sensitivity, reliable detection results, etc., and can simplify the analysis and detection steps, shorten the analysis time, and more importantly make the online real-time detection possible, easy to carry and field work, and has very good application prospect in the field of microbial detection including food safety and rapid detection;
2) the detection method and the biosensor established by the present disclosure can simultaneously realize the dual specific detection of Salmonella and Staphylococcus aureus, the detection has good specificity, high sensitivity, reliable detection result, can be discerned by the naked eye, and the detection process is quick and convenient, which are of great significance in daily monitoring or market screening and other aspects. Specifically, the detection sensitivity of the detection method and the biosensor established by the present disclosure for detecting Salmonella and Staphylococcus aureus are 10 cfu/mL and 10 cfu/mL, respectively; in addition, the specificity experiment results show that the detection method and biosensor established by the present disclosure do not cross-react with Shigella and Escherichia coli, and can realize dual specific detection of Salmonella and Staphylococcus aureus at the same time;
and 3) the color module based on nucleic acid self-assembly according to the present disclosure is a non-enzymatic reaction, the reaction system component is simpler, the reaction process is simpler, the step of terminating the enzymatic reaction is reduced, and the constant temperature reaction simplifies the requirement for the enzymatic reaction temperature, and the economic cost is significantly reduced, the reaction time is shortened to help to meet the requirements of fast and simple detection.

13718731.2 BRIEF DESCRIPTION OF DRAWINGS
[0052] Figure 1 is a schematic diagram showing the structure of ultra-rapid PCR device.
[0053] Figure 2 is the result of verification of the amplification effect of the dual ultra-rapid PCR reaction. Lane 1 is a negative control without product purification (a dual ultra-rapid PCR
reaction system without the addition of genomic samples of Salmonella and Staphylococcus aureus); lane 2 is a positive sample with product purification (a dual ultra-rapid PCR reaction system with the addition of genomic samples of Salmonella and Staphylococcus aureus); lane 3 is a negative control with product purification (a dual ultra-rapid PCR reaction system without the addition of genomic samples of Salmonella and Staphylococcus aureus); and lane 4 is a positive sample with product purification (a dual ultra-rapid PCR reaction system with the addition of genomic samples of Salmonella and Staphylococcus aureus).
[0054] Figure 3 is a standard curve for Salmonella.
[0055] Figure 4 is a standard curve for Staphylococcus aureus.
[0056] Figure 5 are results showing specificity of experiments, wherein 1 is well 1, 2 is well 2, 3 is well 3, 4 is well 4; a is a result of detecting the samples of Salmonella and Shigella; b is a result of detecting the samples of Escherichia coli and Staphylococcus aureus;
and c is a result of detecting the samples of Salmonella and Staphylococcus aureus.
DETAILED DESCRIPTION
[0057] The experimental methods used in the following examples are conventional methods unless otherwise specified.
[0058] The molecular biology experimental methods not specifically described in the following examples are all carried out according to the specific methods listed in the book "Molecular Cloning: A Laboratory Manual" (third edition) by J. Sambrook, or carried out according to the instructions of kits and products.
[0059] The materials and reagents used in the following examples are commercially available 13718731.2 unless otherwise specified.
Example 1: establishment of a dual colorimetric sensing method based on ultra-rapid PCR for detecting Salmonella and Staphylococcus aureus I. Experimental materials [0060] The strain information used in this example is shown in Table 1, the designed nucleotide sequence of primers are shown in Table 2 and Sequence Listing.
Table 1 Bacteria name Latin name Strain number Salmonella Salmonella spp. CGMCC 1.0090 Staphylococcus aureus Staphylococcus aureus ATCC 25923 Table 2 Primer Name Sequence (from 5' to 3') AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol Primer 1 bridge-CTCTCTCTTTCCCTCTCTCTCTCTITTTTGTGAAATTATCGCCA
CGTTCGGGCAA
Primer 2 TCATCGCACCGTCAAAGGAACC
TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol Primer 3 bridge-AACTATACAACCTACTACCTCAAAAAAAAAAAAGCACATAAC
AAGCG
Primer 4 GATAAAGAAGAAACCAGCAG
AGGGCGGGTGGGTCTCTCTCTTTCCCTCTCTCTCTCTCGGCAGAGAGA
Hairpin 1 GAGAGGGAAAGT GGGT
TGGGTAGAGAGAGAGAGGGAAAGAGAGAGCTTTCCCTCTCTCT
Hairpin 2 CTCTGCCGTGGGTAGGGCGGG
AGGGCGGGTGGGTAGTAGGTTGTATAGTTCAAAGTAACTATACAACCT
Hairpin 3 ACTACCTCATGGGT
TGGGTACTTTGAACTATACAACCTACTTGAGGTAGTAGGTTGTATAGTT
Hairpin 4 TGGGTAGGGCGGG
[0061] In Table 2, the nucleotide sequence on the left side of the linking arm 13718731.2 (oxyethyleneglycol bridge) of the upstream Primer 1 is the nucleotide sequence set forth in SEQ
ID NO: 1, and the nucleotide sequence on the right side of the linking arm is the nucleotide sequence set forth in SEQ ID NO: 2, and the chemical structure of the linking arm is:
s 3.
al [0062] In Table 2, the nucleotide sequence of the downstream Primer 2 is the nucleotide sequence set forth in SEQ ID NO: 3.
[0063] In Table 2, the nucleotide sequence on the left side of the linking arm (oxyethyleneglycol bridge) of the upstream Primer 3 is the nucleotide sequence set forth in SEQ
ID NO: 4, and the nucleotide sequence on the right side of the linking arm is the nucleotide sequence set forth in SEQ ID NO: 5, and the chemical structure of the linking arm is the same as the chemical structure of the linking arm of Primer 1.
[0064] In Table 2, the nucleotide sequence of the downstream Primer 4 is the nucleotide sequence set forth in SEQ ID NO: 6.
[0065] In Table 2, each of the hairpin sequences 1 to 4 (Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4) is a G-quadruplex sequence by cleaving two G-quadruplex forming nucleotide sequences at a ratio of 25% and/or 75% and respectively adding to two terminals of the Hairpin Probe, and T
base is added to protect the cleaved G-quadruplex sequences. The two G-quadruplex forming nucleotide sequences are the nucleotide sequence AGGG CGGG TGGG TGGG set forth in SEQ
ID NO: 7 and the nucleotide sequence TGGG TGGG TAGGG CGGG set forth in SEQ ID
NO: 8.
The "initiator" can promote nucleic acid self-assembly, so that the cleaved G-quadruplex get close to each other in distance, and under the induction of hemin, the G-quadruplex functional structure having an activity similar to horseradish peroxidase is formed to catalyze the color development of the reaction between ABTS2- and H202. Specifically, in Table 2, the nucleotide sequence of the hairpin sequence Hairpin 1 is the nucleotide sequence set forth in SEQ ID NO:
9; the nucleotide 13718731.2 sequence of the hairpin sequence Hairpin 2 is the nucleotide sequence set forth in SEQ ID NO: 10;
the nucleotide sequence of the hairpin sequence Hairpin 3 is the nucleotide sequence set forth in SEQ ID NO: 11; and the nucleotide sequence of the hairpin sequence Hairpin 4 is the nucleotide sequence set forth in SEQ ID NO: 12.
[0066] The sequences listed in Table 2 are all artificially synthesized.
[0067] Ex Taq DNA polymerase, lox Ex Taq Buffer (20 mM Mg2+ Plus) and dNTP
Mixture (2.5 mM) are purchased from TAKaRa. Hemin and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS2-) are purchased from Sigma-Aldrich Chemical Co. Experimental water is obtained from Milli-Q water purification system. All other reagents are purchased from China National Pharmaceutical Group Corporation.
II. Construction of ultra-rapid PCR device [0068] The main structure of the ultra-rapid PCR device is shown in Figure 1.
The specific structure, connection method, working principle and working process include:
Light Cycler model capillary (20 I,' 04 929 292 001, Roche) is used as a PCR
sample chamber of the ultra-rapid PCR device, the samples are collected at one terminal of each capillary by rapid centrifugation, and the capillary with the sample is fixed to the plastic holder after centrifuging. The plastic holder is connected to a stepper motor (42JSF630AS-1000, Just Motioin Control) which drives the capillary sample chamber fixed on the plastic holder to switch between a high-temperature water bath at 95 C and a medium-temperature water bath at 58 C cyclically to realize reaction temperature change and control during the ultra-rapid PCR
reaction. The stepper motor is powered by a switching power supply (S-100-24, Elecall), and the frequency or time control of the above-mentioned cyclic conversion of the stepper motor is realized by a DC
servo motor driver (YZ-ACSD60, Moving) and Labview (version 2014). Temperature measurement is achieved by using a thermocouple encapsulated in a capillary.
The amplification and linearization of the thermocouple signal are carried out using a temperature transmitter (SBWR-2260, K, Yuancheng) and processed using the Arduino UNO v1.0 chip. The Arduino UNO chip converts the received analog signal into a digital signal, which is then subjected to 13718731.2 calculation by the Arduino IDE (version 1.8.1) module.
III. Dual ultra-rapid PCR reaction [0069] 1) Preparation of a dual ultra-rapid PCR reaction system, which is shown in Table 3:
Table 3 Reaction component Final concentration Template 0.05 M
Ex Taq DNA polymerase 1.5 U/mL
Primer-1 2 M
Primer-2 2 M
Primer-3 2 M
Primer-4 2 M
dNTP 250 M
10xEx Taq Buffer 1xEx Taq Buffer ddH20 Up to 10 L
[0070] Salmonella and Staphylococcus aureus bacteria were cultured overnight in LB medium for activation, and genomic DNA of Salmonella and Staphylococcus aureus was respectively extracted from the culture by using bacterial genomic DNA extraction kit (New Industry), and 1 pt each of the extracted genomic DNA samples was taken to mix as a template in Table 3.
Primers 1 to 4 (Primer 1, Primer 2, Primer 3, Primer 4) in Table 3 were primers 1 to 4 (Primer 1, Primer 2, Primer 3, Primer 4) listed in Table 2 above.
[0071] 2) Reaction process of the dual ultra-rapid PCR
According to Table 3, 10 I of the reaction system was prepared on ice, and it was quickly placed in the ultra-rapid PCR reaction device constructed in part II for temperature control. The temperature control and cycle number are shown in Table 4:
Table 4 Reaction Reaction time temperature 95 C 4s 30 cycles, 5min in total 13718731.2 58 C 6s [0072] 3) Verification of amplification results for dual ultra-rapid PCR
reactions After the completion of the above dual ultra-rapid PCR reaction, agarose gel electrophoresis with 2% ethidium bromide pre-staining was used to verify the amplification result of the dual ultra-rapid PCR reaction system.
Electrophoresiscondition: 130 V for 25 min Photographing system: Molecular Imager Gel Doc XR (Bio-Rad).
PCR product purification kit (Sangon Biotech) was used to remove primer dimers, unreacted primers and reaction impurities.
[0073] The verification result of the amplification result of the dual ultra-rapid PCR reaction is shown in Figure 2. Figure 2 shows that the dual ultra-rapid PCR reaction system achieves an effective amplification of two types of pathogenic bacteria; and the purification of the PCR
product purification kit effectively removes primer dimers, unreacted primers and reaction impurities.
IV. Establishment of color module based on nucleic acid self-assembly and visual detection of dual pathogen [0074] 1) Sensitivity experiment [0075] Standard curves of Salmonella and Staphylococcus aureus were plotted.
The four hairpin probes listed in Table 2 above: Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4 were dissolved in ultrapure water to 100 M, heated at 95 C for 5 min, and then slowly cooled down to room temperature.
[0076] Salmonella and Staphylococcus aureus bacteria were cultured overnight in LB medium for activation. The culture was subjected to a gradient dilution and then plate culture for counting.
Salmonella or Staphylococcus aureus broth at concentrations of 101 cfu/ml, 102 cfu/ml, 103 cfu/mL, 104 cfu/mL, and 105 cfu/mL were subjected to genomic DNA extraction by using the bacterial genomic DNA extraction kit (New Industry). The genomic DNA samples extracted from 13718731.2 the same concentration of Salmonella broth and Staphylococcus aureus broth were mixed (according to a volume ratio of 1:1, i.e. 1 L of each) as a template, and a dual ultra-rapid PCR
reaction was carried out according to the dual ultra-rapid PCR reaction described in the above part III. The reaction system (10 I) after the completion of the reaction was equally divided into 2 portions: one of which was added with the ultrapure aqueous solutions of Hairpin 1 and Hairpin 2 produced above, and the other was added with the ultrapure aqueous solutions of Hairpin 3 and Hairpin 4 produced above. Self-assembly buffer (8 mM Na2HPO4, 2.5 mM NaH2PO4, 0.15 M
NaCl, 2 mM MgCl2, pH 7.4) was added respectively to the above two portions, and the final concentration of each Hairpin Probe was 2 M, and both reaction systems were 10 I. Both reaction systems were incubated at 37 C for 20 min, and the nucleic acid self-assembly product was obtained.
[0077] The color system based on nucleic acid self-assembly was established.
10 L of nucleic acid self-assembly reaction product was taken and added with 1 L of hemin stock solution (10 M), 32 L G-quadruplex inducing buffer (100 mM 2-(4-morpholine) ethanesulfonic acid (MES), 40 mM KCl, with a volume percentage of 0.05% Triton X-100, pH 5.5), and 23 L
of ultrapure water, the mixture was incubated at 37 C for 20 min; 8 L of ABTS2- stock solution (20 mM) and 8 L of hydrogen peroxide (H202) stock solution (20 mM) were added to the mixture and then incubated at room temperature in the dark for 5 min. After the reaction was completed, the OD value of the reaction solution at 415 nm was measured by a spectrophotometer, and the respective standard curves of Salmonella and Staphylococcus aureus were plotted, and the results are shown in Figure 3 and Figure 4.
[0078] According to the obtained standard curve and the 3,:y principle, the detection limits of Salmonella and Staphylococcus aureus were determined to be 10 cfu/mL and 10 cfu/mL, indicating that the new detection method established by the present disclosure has a high sensitivity.
[0079] The method of plotting the standard curve and determining the detection limit were carried out according to the method described in the literature Macdougall, D., Crummett, W.B., 1980. Anal. Chem. 52(14), 2242-2249.
[0080] 2) Accuracy experiment 13718731.2 [0081] Standard substance recovery test:
[0082] The Salmonella broth with the concentration of 10 cfu/mL and the Staphylococcus aureus broth with the concentration of 10 cfu/mL were respectively detected by a traditional plate detection method and the new method established by the present disclosure, and the detection results are shown in Table 5. The average number of colony detected by the new detection method established by the present disclosure (the detection process was consistent with the sensitivity experiment process described above, and the only difference was that the extracted genomic DNA samples were from Salmonella broth and Staphylococcus aureus broth having a concentration of 10 cfu/mL. After extraction, 1 uL of each sample was taken and mixed as the template) was similar to the average number of colony detected by the traditional plate detection, indicating that the new detection method established by the present disclosure has high accuracy.
Table 5 Plate detection New method Bacteria name Average number of colony Average number of colonya+SDb (cfu/mL) (cfu/mL) Salmonella 9.70 10.30 1.33 Staphylococcus aureus 10.42 9.81 0. 87 [0083] 3) Specificity experiment [0084] Salmonella, Staphylococcus aureus, Shigella and Escherichia colt were cultured overnight in LB medium for activation to prepare 10 cfu/mL of Salmonella broth, 10 cfu/mL of Staphylococcus aureus broth, 100 cfu/ mL of Shigella broth, and 100 cfu/mL of Escherichia coli broth, and genomic DNA samples were extracted from different broth by bacterial genomic DNA
extraction kit (New Industry). Each of the genomic DNA samples extracted from Salmonella broth and Shigella broth was taken 1 1.1L to mix as a template, which was labeled as the reaction system 1; each of the genomic DNA samples extracted from the Escherichia coli broth and Staphylococcus aureus broth was taken 1 L to mix as a template, which was labeled as the reaction system 2; and each of the genomic DNA samples extracted from the Salmonella broth and Staphylococcus aureus broth was taken 1 !IL to mix as a template, which was labeled as the 13718731.2 reaction system 3. Dual ultra-rapid PCR reaction of the three reaction systems was carried out according to the dual ultra-rapid PCR reaction (all the same except the template is replaced accordingly) described above in the above part III.
[0085] The four Hairpin Probes listed in Table 2 above: Hairpin 1, Hairpin 2, Hairpin 3, and Hairpin 4 were dissolved in ultrapure water to 100 M, heated at 95 C for 5 min, and then slowly cooled to room temperature for later use.
[0086] The three reaction systems (10 I) after the completion of the reaction were equally divided into 4 portions. The first portion of each reaction system was added to three wells marked as 1 (the ultrapure aqueous solutions of Hairpin 1 and Hairpin 2 were dissolved in well 1 in advance), and the second portion of each reaction system was added to three wells marked as 2 (the ultrapure aqueous solutions of Hairpin 3 and Hairpin 4 were dissolved in well 2 in advance).
The remaining two portions of each reaction system were added to three wells marked as 3 and three wells marked as 4 respectively (no Hairpin was placed in well 3 and well 4 as a negative control). Then, the self-assembly buffer (8 mM Na2HPO4, 2.5 mM NaH2PO4, 0.15 M
NaC1, 2 mM MgCl2, pH 7.4) was added in each well, and the final concentration of each hairpin probe was 2 M, and each well was 10 L. All the wells were incubated at 37 C for 20 min, and the nucleic acid self-assembly products was obtained.
[0087] 1 'IL of hemin stock solution (10 M), 32 L of G-quadruplex inducing buffer (100 mM
2-(4-morpholine) ethanesulfonic acid (MES), 40 mM KC1, with a volume percentage of 0.05%
Triton X-100, pH 5.5), 23 L of ultrapure water were added to each well to incubate at 37 C for 20 min; and 8 piL of ABTS2- stock solution (20 mM) and 8 L of hydrogen peroxide (H202) stock solution (20 mM) were added to the mixture and incubated at room temperature for 5 min in the dark.
[0088] The experimental results are shown in Figure 5. The detection method established by the present disclosure has no cross-reaction to Shigella and Escherichia coli, and can simultaneously achieve a dual specific detection of Salmonella and Staphylococcus aureus.
[0089] The examples described above are only illustrative of the embodiments of the present disclosure, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the present disclosure. However, any technical solution obtained by using 13718731.2 equivalent replacement or equivalent transformation should fall within the protection scope of the present disclosure.

13718731.2

Claims

1. A detection method, comprising nucleic acid amplification in vitro, wherein the reaction system of the nucleic acid amplification in vitro comprises a upstream primer and a downstream primer, the upstream primer comprises a complementary sequence A, a linking arm, a complementary sequence B, and a nucleotide sequence capable of specifically amplifying a target to be detected, the linking arm is provided between the complementary sequence A and complementary sequence B, and the nucleotide sequence capable of specifically amplifying the target to be detected is provided at 5' terminal or 3' terminal of the upstream primer;
the nucleotide sequence of the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or reverse complementary to each other;
the linking arm comprises a moeity capable of inhibiting polymerase binding and/or a moeity capable of inhibiting new strand extension during in vitro nucleic acid amplification; and the downstream primer comprises the nucleotide sequence capable of specifically amplifying the target to be detected.

2. The method according to claim 1, further comprising at least one of the following 1) to 2):
1) the nucleic acid amplification in vitro includes ultra-rapid PCR, wherein the reaction process of the ultra-rapid PCR comprises: 90-98 °C for 2-6s and 50-60 °C for 2-8s, for 20-40 cycles in total;
2) the linking arm comprises a compound having a long-chain structure.

3. The method according to claim 1 and/or 2, further comprising at least one of the following 1) to 8):
1) the upstream primer includes a primer obtained by linking the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 via the linking arm;
2) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 3;
3) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: I and/or SEQ ID NO: 2 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 1 and/or SEQ ID
NO: 2;
4) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 3 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
3;
5) the upstream primer includes a primer obtained by linking the nucleotide sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5 via the linking arm;
6) the downstream primer includes the nucleotide sequence set forth in SEQ ID
NO: 6;
7) the upstream primer includes a primer obtained by linking, via the linking arm, nucleotide sequences which are modified from the nucleotide sequences set forth in SEQ ID
NO: 4 and/or SEQ ID NO: 5 by substituting, adding and/or deleting one or more nucleotides and have the same function as the nucleotide sequences set forth in SEQ ID NO: 4 and/or SEQ ID
NO: 5; and 8) the downstream primer includes a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 6 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO:
6.

4. A detection method, comprising performing a color reaction based on nucleic acid self-assembly, wherein the reaction system of the color reaction based on nucleic acid self-assembly comprises a hairpin sequence which comprises all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence, a complementary sequence C, and a complementary sequence D, wherein the all or a part of the nucleotide sequence containing a G-quadruplex forming nucleotide sequence is provided at 5' terminal and/or 3' terminal of the hairpin sequence;
the complementary sequence C and the complementary sequence C of the other hairpin sequence are complementary and/or reverse complementary to each other;
and the complementary sequence D and the target to be detected or the nucleotide sequence linked to the target to be detected are complementary and/or reverse complementary.

5. The method according to claim 4, wherein the hairpin sequence comprises at least one of the following 1) to 4):
1) the nucleotide sequence set forth in SEQ ID NO: 9 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 9 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 9;
2) the nucleotide sequence set forth in SEQ ID NO: 10 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 10 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 10;
3) the nucleotide sequence set forth in SEQ ID NO: 11 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 11 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 11; and 4) the nucleotide sequence set forth in SEQ ID NO: 12 and/or a nucleotide sequence which is modified from the nucleotide sequence set forth in SEQ ID NO: 12 by substituting, adding and/or deleting one or more nucleotides and has the same function as the nucleotide sequence set forth in SEQ ID NO: 12.

6. The method according to claim 4 and/or 5, wherein the color reaction based on nucleic acid self-assembly further comprises at least one of the following 1) to 2):
1) the reaction condition of the color reaction based on nucleic acid self-assembly comprises a step of incubating at 37 C for 20 min; and 2) the final concentration of the hairpin sequence in the reaction system of the color reaction based on nucleic acid self-assembly is 2p.M.

7. A detection method, comprising first amplifying a target to be detected by the method according to claims 1, 2 and/or 3, and then detecting the target by the method according to claims 4, 5 and/or 6; and specifically, the complementary sequence D according to claim 4 and the complementary sequence A and/or the complementary sequence B according to claim 1 are complementary or reverse complementary.

8. The method according to claim 7, further comprising at least one of the following 1) to 4):
1) determining whether an object to be detected contains the target to be detected by a color change of final reaction system;
2) calculating the concentration of the target to be detected in the object by a method of plotting standard curve according to the color of the final reaction system;
3) amplifying the detection signal of the target to be detected by increasing the type of hairpin sequence in the reaction system; and 4) achieving dual or multiple detection by increasing the type of upstream primer or downstream primer, and simultaneously increasing the type of hairpin sequence in the reaction system.

9. A kit and/or a biosensor, comprising at least one of the following 1) to 2):
1) a upstream primer and a downstream primer, the upstream primer comprises a complementary sequence A, a linking arm, a complementary sequence B, and a nucleotide sequence capable of specifically amplifying a target to be detected, wherein the linking arm is provided between the complementary sequence A and complementary sequence B, and the nucleotide sequence capable of specifically amplifying the target to be detected is provided at 5' terminal or 3' terminal of the upstream primer;
the nucleotide sequence of the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or reverse complementary to each other;
the linking arm comprises a moeity capable of inhibiting polymerase binding and/or a moeity capable of inhibiting new strand extension during in vitro nucleic acid amplification; and the downstream primer comprises the nucleotide sequence capable of specifically amplifying the target to be detected; and 2) a hairpin sequence comprises all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence, a complementary sequence C, and a complementary sequence D, wherein the all or a part of a nucleotide sequence containing a G-quadruplex forming nucleotide sequence is provided at 5' terminal and/or 3' terminal of the hairpin sequence;
the complementary sequence C and the complementary sequence C of the other hairpin sequence are complementary and/or reverse complementary; and the complementary sequence D and the target to be detected or the nucleotide sequence linked to the target to be detected are complementary and/or reverse complementary.

10. Use of the method according to claims 1, 2 and/or 3, the method according to claims 4 and/or 5, the method according to claims 6 and/or 7, and the kit and/or the biosensor according to claim 8 and/or 9.