RPA-CRISPR / Cas12a primer sets, gRNA and probes, kits and detection systems

Abstract

The present invention belongs to the biological field, and discloses an RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting intracellular Lawsonia, including an RPA primer set and a probe; the RPA primer set includes an upstream primer and a downstream primer as shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of the gRNA is shown in SEQ ID NO.3, the sequence of the probe is shown in SEQ ID NO.4, and the 5-end and 3-end of the probe are fluorescent groups and quenching groups, respectively. It has the advantages of low detection limit and high detection accuracy. At the same time, the present invention also discloses a kit and a detection system for intracellular Lawsonia.

Description

Technical Field

[0001] The present invention relates to the biological field, and specifically to an RPA-CRISPR / Cas12a primer set, gRNA and probe, a kit and a detection system for detecting intracellular Lawsonia spp. Background Art

[0002] Lawsonia intraculularis (LI) is an intestinal pathogen that causes porcine proliferative enteritis (PPE). It mainly colonizes the terminal ileum. When the disease is severe, it can also infect the jejunum and colon. The bacteria are mostly Campylobacter parvus, which is Gram-negative and acid-fast positive. LI infection is a bacterial infectious disease of pigs. The disease has a strong ability to spread. Once sick pigs appear in a pig farm, a large number of pigs can be infected. The sick pigs have a decreased appetite, slow growth and development, and a reduced feed-to-meat ratio, which seriously threatens the healthy development of the world's pig industry.

[0003] At present, the existing methods for detecting LI have certain limitations, such as Giemsa staining, Gram staining and microscopic examination, which can only be used for the preliminary identification of LI; histopathological sections and immunohistochemistry are mostly used for postmortem detection of pigs; PCR method can directly detect pathogenic nucleic acid to determine whether pigs are infected with LI, but in some chronic or subclinical cases, there is intermittent bacterial excretion, and the positive rate of PCR detection is different at different sampling times, which can easily cause false negative test results; enzyme-linked immunosorbent assay (ELISA) has many interfering factors, and the control of temperature and time may affect the experimental results, resulting in false positives; IPMA and IFA are the most specific methods for detecting LI antibodies, but both methods require in vitro culture of LI-infected cells, and conventional methods are difficult to culture LI, and there is no report on the successful isolation and culture of LI in China, which has become a major difficulty in the detection of LI. Compared with the existing detection methods, the RPA-CRISPR / Cas12a detection method established in this study has a low detection limit, high detection accuracy, simple operation, no need for expensive equipment, and diverse application scenarios, providing a practical technical means for rapid detection and epidemiological investigation of LI.

[0004] Recombinase polymerase amplification (RPA) is a highly sensitive isothermal amplification technology that exponentially amplifies the target through recombinase, polymerase, single-stranded binding protein, and primers. The reaction conditions are 37℃-42℃, and detectable amplification products can be obtained in about 15 to 30 minutes. Due to the mild reaction conditions, high amplification efficiency, and simple equipment of RPA, it has been widely used in the detection of pathogens in recent years. This also provides a new option for on-site detection and is called a nucleic acid detection technology that can replace PCR.

[0005] The CRISPR / Cas system, whose full name is clustered regularly interspaced palindromic repeats / CRISPR-associated proteins, is a third-generation genome editing tool derived from bacterial or archaeal acquired immunity, a technology that guides Cas proteins to modify targeted sequences by RNA. The CRISPR / Cas system consists of four parts, namely CRISPR clusters, leader sequences, repeat sequences, and a series of conservative CRISPR-related genes. Compared with other cas proteins, the target DNA of Cas12a is no longer limited to double-stranded DNA (dsDNA), but can also efficiently cut single-stranded DNA (ssDNA). According to this characteristic, Cas12a is applied to pathogen detection, and the detected system mainly includes three parts: target sequence, Cas12a-sgRNA complex, and fluorescent reporter, in which the two ends of the fluorescent reporter molecule are fluorescent groups and quenching groups, connected by single-stranded DNA, at which time the quenching group can inhibit the luminescence of the fluorescent group, and the system cannot observe fluorescence. When sgRNA recognizes the target sequence, it can activate the trans-cleavage activity of Cas12a, and the Cas12a protein begins to non-specifically cut the DNA around it, including the fluorescent reporter. After the single-stranded DNA connected to the fluorescent reporter is cut, the quenching group will not be able to inhibit the luminescence of the fluorescent group. At this time, the system can be observed to produce fluorescence, thereby identifying whether there is a target sequence to be detected in the system.

[0006] The RPA-CRISPR / Cas12a rapid detection method is very difficult when designing primers, probes, and crRNA. The most core difficulty lies in the selection of sites and the design of crRNA and RPA primer sets.

[0007] 1. The difference in conserved sites will lead to huge differences in test results; this difference will not be reflected in ordinary PCR detection, because ordinary PCR technology can generally obtain corresponding test results and meet the required sensitivity as long as the corresponding sequence is amplified; however, RPA-CRISPR / Cas12a technology involves the formation of a ternary complex between the target sequence and crRNA and Cas12a. The non-specific cleavage activity of Cas12a protein is activated when the target sequence forms a ternary complex with crRNA and Cas12a, thereby cutting the test strip probe and fluorescent probe in the system to generate a fluorescent signal or the test strip detection line appears; this not only puts forward requirements for the efficiency of primer amplification and the specificity of crRNA, but also puts forward more important requirements for the selection of sites.

[0008] 2. Primer sequence. Primers are used to extend the sequence of the conserved site. For the same site, different primers are used for amplification. Even if the obtained sequences are slightly different, the final sensitivity and specificity may be significantly different. This is because the target sequence and crRNA, Cas12a form a ternary complex, which affects the cutting and display results.

[0009] 3. crRNA. Since the target sequence, crRNA and Cas12a form a ternary complex, existing studies have found that suitable crRNA is particularly important for specificity and sensitivity.

[0010] At the same time, unlike PCR technology, there is no mature software to design

[0011] The relevant sequences in the RPA-CRISPR / Cas12a rapid detection method require researchers to obtain relevant sequences based on long-term research and exploration;

[0012] In summary, it is challenging to successfully develop RPA-CRISPR / Cas12a rapid detection methods and systems. Summary of the invention

[0013] The first object of the present invention is to provide an RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting intracellular Lawsonia spp., which have the advantages of low detection limit and high detection accuracy.

[0014] Meanwhile, the invention also discloses a kit and a detection system of intracellular Lawsonia spp.

[0015] To achieve the above first purpose, the present invention provides the following technical solutions:

[0016] An RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting intracellular Lawsonia spp., comprising an RPA primer set, gRNA and probe;

[0017] The RPA primer set includes an upstream primer and a downstream primer as described in SEQ ID NO.1;

[0018] The sequence of gRNA is shown in SEQ ID NO.2;

[0019] The sequence of the probe is 5'-FAM-TTATT-BHQ1-3';

[0020] The 5' end and 3' end of the probe are respectively a fluorescent group and a quenching group.

[0021] As shown in Table 1 below:

[0022] Table 1 Design of primers, probes and gRNA for RPA-CRISPR / Cas12a detection of Lawsonia intracellularis

[0023]

[0024] The present invention also discloses a kit comprising the RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting intracellular Lawsonia spp. as described above.

[0025] At the same time, the present invention also discloses a detection system for detecting intracellular Lawsonia, including an amplification system and a CRISPR / Cas12a reaction system;

[0026] The amplification system is:

[0027] 2.4 μL of the upstream primer at a concentration of 10 μmol / L;

[0028] 2.4 μL of downstream primer at a concentration of 10 μmol / L;

[0029] Primer Free Rehydration Buffer 29.5μL;

[0030] Deionized water 11.2 μL;

[0031] Mix the above substances and add them into the freeze-dried tube, mix them by blowing, and divide them into two.

[0032] Add 1 μL of DNA template to each tube, mix well, and then add 1.25 μL of 280 mmol / L MgoAc to each tube;

[0033] The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0034] The CRISPR / Cas12a reaction system is:

[0035] Deionized water 13 μL

[0036] 1 μL of Cas12a enzyme at a concentration of 1 μmol / L;

[0037] 1 μL of gRNA at a concentration of 1 μmol / L;

[0038] 10x units of NEB buffer 2.1 2 μL;

[0039] 1 μL of ssDNA reporter molecule with a concentration of 10 μmol / L;

[0040] 2 μL of nucleic acid amplification product obtained by amplification of the amplification system;

[0041] The gRNA sequence is shown in SEQ ID NO.3.

[0042] The beneficial effects of the present invention are as follows:

[0043] The RPA-CRISPR / Cas12a system of the present invention has high specificity for the detection of intracellular Lawsoniae, and has the advantages of low detection limit and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0045] Figure 1 Design pattern diagram of primers and gRNA used in this experiment

[0046] Figure 2A It is a graph of fluorescence intensity at different SSDNA concentrations;

[0047] Figure 2B Fluorescence intensity photos at different SSDNA concentrations;

[0048] Figure 3 Fluorescence photos during the optimization of Cas12a protein concentration and gRNA concentration;

[0049] Figure 4A This is the test result table of different samples;

[0050] Figure 4B The test results of different samples are shown in Figure 2.

[0051] Figure 5A It is a graph of fluorescence values ​​of the experimental group and the control group without template;

[0052] Figure 5B Fluorescence photos of the experimental group and the control group without template;

[0053] Figure 5CThis is the test result diagram of common PCR;

[0054] Fig. 6A The following are photos of the test results of different target genes;

[0055] Figure 6B It is a graph of fluorescence values ​​of different target genes;

[0056] Fig. 7A Table of fluorescence values ​​for combination experiments with different primers and gRNA3;

[0057] Figure 7B The results of the combination experiment of different primers and gRNA3 are shown in the following figure;

[0058] Fig. 8A Table of fluorescence values ​​when testing F4R4 and different gRNA combinations;

[0059] Figure 8B Fluorescence photos of the detection of F4R4 and different gRNA combinations. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0062] The clinical sample nucleic acids used in the present invention were all obtained from the Swine Disease Laboratory of the Animal Health Institute of Guangdong Academy of Agricultural Sciences.

[0063] The materials used in the following examples and test examples include:

[0064] The Lawsonia intracellularis primer set, probe and Lawsonia intracellularis recombinant plasmid standard of the present invention were synthesized by Shanghai Sangon Biotechnology Co., Ltd.;

[0065] Basic was purchased from TwistDx, UK;

[0066] EnGen Lba Cas12a was purchased from NEB, USA

[0067] gRNA was synthesized by Huzhou Hippo Biotechnology Co., Ltd.;

[0068] Fecal genomic DNA / RNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.;

[0069] LC480 Roche real-time fluorescence quantitative PCR instrument was purchased from ROCHE.

[0070] Example 1

[0071] RPA primers were designed based on the pcm gene of Lawsonia intracellularis in GenBank. The software was used to find the protospacer adjacent motif (PAM) in the RPA amplified product. The gRNA that was complementary to the target sequence was designed based on the position of the PAM sequence. After activation, Cas12a can not only cleave the target DNA, but also has an auxiliary cleavage activity that can cleave any single-stranded DNA, so an ssDNA reporter molecule needs to be designed. Primers, probes, and gRNA refer to Table 1. Specific design ideas can be referred to Figure 1 .

[0072] Example 2

[0073] Optimization of amplification conditions

[0074] 1. Optimization of ssDNA reporter concentration

[0075] The ssDNA reporter molecules were optimized according to the final concentrations of 10umol / L, 8umol / L, 6umol / L, 4umol / L, 2umol / L, and 1umol / L, and the fluorescence intensity was observed ( Figure 2A Figure 2B ), the optimal concentration is 8umol / L.

[0076] Figure 2A It is a graph of fluorescence intensity at different SSDNA concentrations;

[0077] Figure 2B Fluorescence intensity photos at different SSDNA concentrations;

[0078] Figure 2B In the figure, from left to right they are 10umol / L, 8umol / L, 6umol / L, 4umol / L, 2umol / L, and 1umol / L.

[0079] 2. Optimization of Cas12a protein concentration and gRNA concentration

[0080] Cas12a protease and gRNA were optimized according to the final concentration of 1umol / L, 800nmol / L, 600nmol / L, and 400nmol / L. The two were subjected to orthogonal experiments. The fluorescence values ​​of 465-510nm wavelength and 37℃ for 30min were collected using a real-time fluorescence quantitative PCR instrument. The fluorescence values ​​were shown in Table 2 and Table 3. Figure 3 ), the fluorescence value was maximum when the concentration of Cas12a protease and gRNA was 1umol / L.

[0081] Table 2 Fluorescence values ​​at different Cas12a protease and gRNA concentrations

[0082]

[0083] Figure 3 Fluorescence photos during the optimization of Cas12a protein concentration and gRNA concentration;

[0084] Figure 3 The meaning of each symbol is:

[0085] The concentration of 1-4gRNA was 1umol / L, and the concentration of Cas12a was 1umol / L, 800nmol / L, 600nmol / L, and 400nmol / L respectively;

[0086] The concentration of 5-8gRNA was 800nmol / L, and the concentration of Cas12a was 1umol / L, 800nmol / L, 600nmol / L, and 400nmol / L, respectively;

[0087] The concentration of 8-12gRNA was 600nmol / L, and the concentration of Cas12a was 1umol / L, 800nmol / L, 600nmol / L, and 400nmol / L respectively;

[0088] The concentration of 12-16gRNA was 400nmol / L, and the concentration of Cas12a was 1umol / L, 800nmol / L, 600nmol / L, and 400nmol / L, respectively;

[0089] N is the negative control.

[0090] Example 3

[0091] Specificity test

[0092] Using LIDNA template as positive control and ddH2O as negative control, the RPA-Cas12a detection method established in this study was used to detect the genomes of Escherichia coli, Streptococcus, Toxoplasma gondii, Clostridium perfringens, PDCoV, TGEV, PCV, and PEDV, respectively. Water was used as negative control. Three replicates were set for each sample. The fluorescence value of the test results was calculated based on the fluorescence value of the test results. Figure 4A and 4B ) Whether the detection method established by this institute is specific.

[0093] Figure 4A This is the test result table of different samples;

[0094] Figure 4B The test results of different samples are shown in Figure 2.

[0095] Results: Reference Figure 4B , only the LIDNA template showed an obvious fluorescence reaction, while Escherichia coli, Streptococcus, Toxoplasma gondii, Clostridium perfringens, PDCoV, TGEV, PCV, and PEDV showed no fluorescence reaction. Figure 4A The fluorescence values ​​of each genome are shown, and it can be seen that the fluorescence value of the LIDNA template is the most significant.

[0096] Figure 4B In the figure, from left to right: Escherichia coli, PDCoV, TGEV, Clostridium perfringens, Toxoplasma gondii, PEDV, PCV, Streptococcus, LI, and negative control.

[0097] Taking Lawsonia intracellularis as an example, the detection steps of the present invention are specifically described as follows: The reagents used in the present invention are as follows: RPA amplification kit Twist Basic, primers, Cas12a protein, NEB buffer 2.1, gRNA, ssDNA reporter molecule, ddH2O; the specific experimental steps of the present invention are as follows:

[0098] Step 1: Extract DNA from the sample to be tested;

[0099] Step 2: Performing a nucleic acid amplification reaction on the DNA obtained in step 1 using an RPA primer set and an RPA amplification kit to obtain a nucleic acid amplification product;

[0100] Step 3: Mix the gRNA, probe and Cas12a enzyme with the nucleic acid amplification product obtained in step 2, and perform CRISPR / Cas12a reaction on the nucleic acid amplification product;

[0101] Step 4: Determine the test result through fluorescence detection.

[0102] In the above method, the system of step 2 is:

[0103] Primer Free Rehydration Buffer 29.5μL;

[0104] 2.4 μL of the upstream primer at a concentration of 10 μmol / L;

[0105] 2.4 μL of downstream primer at a concentration of 10 μmol / L;

[0106] Deionized water 11.2 μL;

[0107] Mix the above substances and add them into the freeze-dried tube, mix them by pipetting, and divide it into two;

[0108] Add 1 μL of DNA template to each tube, mix well and add 1.25 μL of MgoAc with a concentration of 280 mmol / L to each tube.

[0109] The system of step 3 is:

[0110] 10x units of NEB buffer 2.1 2 μL;

[0111] 1 μL of Cas12a enzyme at a concentration of 1 μmol / L;

[0112] 1 μL of gRNA at a concentration of 1 μmol / L;

[0113] 1 μL of ssDNA reporter molecule with a concentration of 10 μmol / L;

[0114] 2 μL of nucleic acid amplification product from step 2

[0115] Deionized water 13 μL

[0116] The reaction condition of step 2 is 39° C. for 20 min; the reaction condition of step 3 is 37° C. for 30 min, and the fluorescence signal is observed.

[0117] The above experiments can confirm that the intracellular Lawsonia RPA-CRISPR / Cas12a detection method has good accuracy and specificity. Finally, through repeated verification, the optimal combination of primers and gRNA, as well as probe, gRNA, and Cas12a concentrations were screened to achieve the purpose of the present invention.

[0118] Example 4

[0119] Sensitivity test

[0120] After calculating the copy number of the recombinant plasmid standard, ten-fold serial dilutions were performed to 10 10 copies / μL, 10 9 copies / μL, 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0The detection method established by this institute was used for detection, ddH2O was set as a negative control, and the LI identification method (PCR) routinely used in this laboratory was used for detection to compare the sensitivity difference between the detection method established by this test and the PCR detection method.

[0121] Results: Reference Figure 5A to Figure 5C ;

[0122] Figure 5A It is a graph of fluorescence values ​​of the experimental group and the control group without template;

[0123] Figure 5B Fluorescence photos of the experimental group and the control group without template;

[0124] Figure 5C This is the test result diagram of common PCR;

[0125] After using the RPA-Cas12a detection method, when 10 0 ~10 4 When the target gene with 10 copies / μL was involved in the reaction, the fluorescence values ​​of the experimental group and the control group without template were significantly different (P<0.001) ( Figure 5A ). Visual observation showed that each reaction in the experimental group had an obvious fluorescent signal, and was significantly different from the no-template control group ( Figure 5B ). The results show that the sensitivity of this detection method can be as low as a single copy. The sensitivity of ordinary PCR is only 10 4 copies / μL( Figure 5C ).

[0126] Comparative Example 1

[0127] The detection target gene of intracellular Lawsoniae of the present invention is the pcm gene; the gene sequence was downloaded from GenBank for sequence alignment analysis, which showed that the gene was well conserved. At the same time, we also screened two other relatively conservative gene groups, ubie and aspA, for experiments, and designed primers for RPA of the three target genes in Primer Premier 5.0. The parameters were set on the benchling: the gRNA length was 20bp, the PAM sequence was TTTN (N is an arbitrary nucleotide), and 3 gRNAs corresponding to the three target genes that met the above conditions and had a score greater than 95 were selected. Referring to the above detection method, the primers, gRNAs, and probes used are shown in Table 3 below:

[0128] Table 3 Best target gene screening table

[0129]

[0130] The test results can be referred to Fig. 6A and Figure 6B ;

[0131] Fig. 6A The following are photos of the test results of different target genes;

[0132] Figure 6B It is a graph of fluorescence values ​​of different target genes;

[0133] The purpose of this comparative example is to screen out the most suitable target gene. According to the above detection method, three target genes were screened. In the same detection system, pcm showed a more efficient cutting efficiency compared with the other two genes ( Fig. 6A and 6B ). Among them, the cleavage efficiency of aspA was the most unstable among the three genes.

[0134] Comparative Example 2

[0135] From Example 1, pcm was selected as the target gene of this experiment. RPA primers (4 pairs) were designed according to the pcm gene sequence of Lawsonia intracellularis in GenBank, and the experiments were combined with gRNA3 respectively. Referring to the above detection method, the primers, gRNA, and probe used are shown in Table 4 below:

[0136] Table 4 Best primer screening table

[0137]

[0138] Results Reference Fig. 7A and Figure 7B ;

[0139] Fig. 7A Table of fluorescence values ​​for combination experiments with different primers and gRNA3;

[0140] Figure 7B The results of the combination experiment of different primers and gRNA3 are shown in the following figure;

[0141] The selection of suitable primers plays a vital role in this detection method. Therefore, in order to screen out the most suitable primers, this comparative example uses the above detection method to perform a combination experiment with the primers and gRNA3, and finally screens out F4R4 as the best primer ( Fig. 7A and Figure 7B ).

[0142] Comparative Example 3

[0143] The best primer F4R4 was screened out from Comparative Example 2, but the selection of gRNA is also particularly important, so the software was used to find the prototype spacer adjacent motif (PAM) in the RPA amplified product, and the gRNA (3) complementary to the target sequence base was designed according to the PAM sequence position. The best primer was combined with the 3 gRNAs to screen the best gRNA. The primers, gRNAs, and probes used are shown in Table 5 below;

[0144] Table 5 Optimal gRNA screening

[0145]

[0146]

[0147] Results Reference Fig. 8A and Figure 8B ;

[0148] Fig. 8A Table of fluorescence values ​​when testing F4R4 and different gRNA combinations;

[0149] Figure 8B Fluorescence photos of the detection of F4R4 and different gRNA combinations.

[0150] According to the fluorescence value, gRNA3 was selected as the best gRNA ( Fig. 8A and 8B ).

[0151] Result analysis:

[0152] 1. It can be seen from Examples 1 to 4 that the method of the present invention has high specificity, sensitivity and accuracy;

[0153] 2. It can be seen from Comparative Example 1 that there are obvious differences in cutting efficiency when using different conservative gene sequences. The outstanding contribution of the present invention is not only to use a new and unrecognized site, but also to verify that this site has better cutting effect and detection results than other conservative sites.

[0154] 3. It can be seen from Comparative Examples 2 and 3 that for the RPA-CRISPR / Cas12a detection system, the selection of appropriate RPA primer sets, probes, and gRNAs is very random. In order to obtain low detection limits and high accuracy results, a large number of combined experiments are required; Comparative Examples 2 and 3 only show part of the experimental results of the present invention, and a large number of failed results are not shown.

[0155] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. An RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting Lawsonia intracellularis, characterized in that: It comprises an RPA primer set, a gRNA and a probe; the RPA primer set comprises an upstream primer and a downstream primer as shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of the gRNA is shown in SEQ ID NO.3; the structure of the sequence of the probe is 5'-FAM-TTATT-BHQ1-3'; the fluorescent group of the probe is FAM, and the quenching group of the probe is BHQ1.

2. A kit, characterized in that: Contains the RPA-CRISPR / Cas12a primer set, gRNA and probe for detecting intracellular Lawsonia spp. as claimed in claim 1.

3. A detection system for detecting Lawsonia intracellularis, characterized in that: Including amplification system and CRISPR / Cas12a reaction system; The amplification system is: 2.4 μL of the upstream primer at a concentration of 10 μmol / L; 2.4 μL of downstream primer at a concentration of 10 μmol / L; Primer Free Rehydration Buffer 29.5 μL; Deionized water 11.2 μL; Mix the above substances and add them into the freeze-dried tube, mix them by blowing, and divide them into two. Add 1 μL of DNA template to each tube, mix well, and add 1.25 μL of 280 mmol / L MgOAc to each tube; The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and SEQ ID NO.2; The CRISPR / Cas12a reaction system is: Deionized water 13 μL; 1 μL of Cas12a enzyme at a concentration of 1 μmol / L; 1 μL of gRNA at a concentration of 1 μmol / L; 10x units of NEB buffer 2.1 2 μL; 1 μL of ssDNA reporter molecule with a concentration of 10 μmol / L; 2 μL of nucleic acid amplification product obtained by amplification of the amplification system; The gRNA sequence is shown in SEQ ID NO.3; The sequence of the ssDNA reporter molecule is 5'-FAM-TTATT-BHQ1-3'.

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