阅读说明：本技术 一种CRISPR/Cas12a一步核酸检测方法 (CRISPR/Cas12a one-step nucleic acid detection method ) 是由 田亚晨 刘箐 刘程 刘涛 方水琴 于 2020-12-30 设计创作，主要内容包括：本发明提供了一种CRISPR/Cas12a一步核酸检测方法,包括以下步骤：步骤1,选取待检测病菌的靶标序列,设计RPA上下游引物并进行特异性筛选后扩增靶标序列,得到引物扩增子序列,利用NCBI BLAST对引物扩增子序列进行特异性验证；步骤2,设计特异性的crRNA；步骤3,设计单链DNA报告分子；步骤4,将单链DNA报告分子、RPA上下游引物、冻干RPA反应颗粒、醋酸镁、RPA水化缓冲液、NEB buffer2.1、RNase Inhibitor与待测靶标进行混合,得到反应混合物；步骤5,构建包括反应物放置管以及注射器的一步检测试剂盒,将反应混合物置于反应物放置管底部,将crRNA与Cas12a蛋白预装到注射器；步骤6,在反应混合物反应后,推动注射器的活塞使得crRNA和Cas12a蛋白与反应混合物混合进行反应,再通过蓝光激发,得到检测结果。(The invention provides a CRISPR/Cas12a one-step nucleic acid detection method, which comprises the following steps: step 1, selecting a target sequence of pathogenic bacteria to be detected, designing an RPA upstream primer and a downstream primer, carrying out specificity screening, amplifying the target sequence to obtain a primer amplicon sequence, and carrying out specificity verification on the primer amplicon sequence by using NCBI BLAST; step 2, designing specific crRNA; step 3, designing a single-stranded DNA reporter molecule; step 4, mixing single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer, NEB buffer2.1 and RNase Inhibitor with a target to be detected to obtain a reaction mixture; step 5, constructing a one-step detection kit comprising a reactant placing tube and a syringe, placing a reaction mixture at the bottom of the reactant placing tube, and pre-loading crRNA and Cas12a protein into the syringe; and step 6, after the reaction mixture reacts, pushing a piston of the injector to mix the crRNA and the Cas12a protein with the reaction mixture for reaction, and then exciting by blue light to obtain a detection result.)

1. A CRISPR/Cas12a one-step nucleic acid detection method is characterized by comprising the following steps:

step 1, selecting a target sequence of pathogenic bacteria to be detected, designing and synthesizing RPA upstream and downstream primers for amplifying the target sequence, carrying out specificity screening, amplifying the target sequence by using the RPA upstream and downstream primers after the specificity screening to obtain a primer amplicon sequence, and carrying out specificity verification on the primer amplicon sequence by using NCBIBLAST;

step 2, designing and detecting the required specific crRNA according to the target sequence;

step 3, designing single-stranded DNA reporter molecules for fluorescence signal detection and blue light excitation visual detection;

step 4, mixing the single-stranded DNA reporter molecule, the RPA upstream and downstream primers, the freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1, RNase Inhibitor and a target to be detected to obtain a reaction mixture;

step 5, constructing a one-step detection kit comprising a reactant placing tube and a syringe arranged in the reactant placing tube, then placing the reaction mixture at the bottom of the reactant placing tube, mixing the crRNA and the Cas12a protein, and pre-loading the mixture into the syringe;

step 6, after the reaction mixture reacts, pushing the piston of the injector to mix and react the crRNA and the Cas12a protein with the reaction mixture, and then obtaining a detection result through blue light excitation,

when the crRNA is designed, the cleavage site of the crRNA on the target sequence and the complementary strand of the crRNA is positioned on the primer amplicon sequence, and then according to the designed sequence of the crRNA, chemical synthesis is directly carried out or a crRNA in-vitro transcription vector is constructed and in-vitro transcription and purification are carried out by using a T7 transcription kit.

2. The one-step nucleic acid detection method for CRISPR/Cas12a according to claim 1, wherein:

wherein, the specific extraction process of the target to be detected in the step 4 comprises the following substeps:

step 4-1, taking 1mL of overnight-cultured bacterial liquid of the bacteria to be detected, centrifuging for 5min at the speed of 8000r/min, and then discarding the supernatant to obtain bacteria;

and 4-2, washing and coating the thallus twice with 500ul of physiological saline, then re-suspending the thallus by using a TE buffer solution with the pH value of 7.6, boiling for 10min, immediately freezing for 5min at the temperature of-20 ℃, centrifuging for 5min at the speed of 12000r/min, and taking the supernatant as the target to be detected for later use.

3. The one-step nucleic acid detection method for CRISPR/Cas12a according to claim 1, wherein:

wherein the step 6 comprises the following substeps:

step 6-1, placing the reaction mixture in a constant temperature detector at 37 ℃ for real-time signal monitoring, wherein the reaction time is 5-10min, and obtaining the reaction mixture after reaction;

and 6-2, pushing a piston of the injector to mix the crRNA, the Cas12a protein and the reacted reaction mixture, continuing to react at 37 ℃ for 40min, exciting by using blue light, and observing by naked eyes to obtain a detection result.

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a CRISPR/Cas12a one-step nucleic acid detection method.

Background

Listeria Monocytogenes (LM) is an important food-borne pathogen which can penetrate intestinal barrier, placenta barrier and blood brain barrier, has the death rate of 20-30 percent and seriously harms the safety and health of human beings. The pathogenicity of the listeria monocytogenes is closely related to virulence factors, and the rapid and accurate identification of the listeria monocytogenes related virulence genes can provide important reference for researching the pathogenicity of the listeria monocytogenes.

At present, the main method for detecting the gene level is to realize the amplification of a target gene through a polymerase chain reaction, and the visualization of an amplification product is realized through agarose gel electrophoresis. Quantitative PCR or fluorescent real-time PCR adopts TaqMan probe and fluorescent dye to carry out real-time detection on the instrument. However, the method depends on precise temperature control equipment, so that the cost is high, the detection steps are complex, and the result cannot meet the requirements of field detection. Especially, RPA is receiving more and more attention due to its characteristics of high sensitivity, easy operation, etc. RPA is an isothermal amplification method, which uses thermostable DNA polymerase to amplify at a constant temperature (37-48 ℃), does not need complex instruments, and can realize instant detection.

CRISPR (CRISPR) is used as an acquired immune system for archaea and most bacteria, and when a virus invades, the bacteria generate corresponding crRNA capable of recognizing viral genome, and the crRNA guides Cas protein (CRISPR-associated proteins) with endonuclease activity to specifically recognize and cut a viral target sequence.

In 2018, based on the discovery of the 'additionally cleavage' activity of Cas13a effector protein, Jonathan S.Goodenbey et al developed a detection platform SHERLOCK (Specific High-Sensitivity enzyme Reporter Unlocking) based on LwCas13a to detect Zika virus, Dengue virus and pathogenic bacteria. In the SHERLOCK system, firstly, a Recombinase Polymerase Amplification (RPA) is used for amplifying a target, after an amplified product is added into the CRISPR/Cas13a system, crRNA specifically recognizes a target nucleic acid sequence so as to excite the accessory cleavage function of Cas13a, and then ssRNA-FQ in the cleavage system generates a detectable fluorescent signal.

The v-form Cas12a, which also has "nicking" activity, can also be applied in the field of nucleic acid detection. In the LbCas12a, HOLMES (an one-Hour Low-cost Multipurpose high yield Efficient System) is developed by Li, wherein the HOLMES combines the CRISPR/Cas12a System with the PCR amplification technology, the amplified product is added into the CRISPR/Cas12a System, the crRNA can specifically recognize the dsDNA amplicon, the attached cleavage function of the Cas12a is excited, and the ssDNA-FQ in the cleavage System generates a detectable fluorescent signal (figure 2). The whole detection system can complete the detection of the target DNA within 1h, and the HOLMES can detect aM DNA virus and RNA virus and accurately distinguish the genotype of the virus and the single nucleotide polymorphism SPN of the human. In 2018, the Doudna team combines the CRISPR/Cas12a system with the RPA isothermal amplification technology, and develops a nucleic acid detection technology DETECTOR (DNA Endonuclease Targeted CRISPR Trans reporter) capable of realizing aM sensitivity. The detection system can be used for detecting Human Papilloma Virus (HPV) in clinical samples within 70min, and accurately judging the types of infected HPV (HPV16 and HPV 18).

The above detection technology based on CRISPR is generally divided into two steps, the first step is to amplify a target gene, and the second step is to detect the target gene by using CRISPR. Although the method has high sensitivity, the detection time is prolonged, the cover opening operation is still required, aerosol pollution is easily caused, false positive is generated, and the method is not beneficial to practical application.

Disclosure of Invention

The invention is carried out to solve the problems and aims to provide a CRISPR/Cas12a one-step nucleic acid detection method.

The invention provides a CRISPR/Cas12a one-step nucleic acid detection method, which is characterized by comprising the following steps: step 1, selecting a target sequence of pathogenic bacteria to be detected, designing and synthesizing RPA upstream and downstream primers for amplifying the target sequence, carrying out specificity screening, amplifying the target sequence by using the RPA upstream and downstream primers subjected to the specificity screening to obtain a primer amplicon sequence, and carrying out specificity verification on the primer amplicon sequence by using NCBI BLAST; step 2, designing and detecting the required specific crRNA according to the target sequence; step 3, designing single-stranded DNA reporter molecules for fluorescence signal detection and blue light excitation visual detection; step 4, mixing single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer, NEB buffer2.1 and RNase Inhibitor with a target to be detected to obtain a reaction mixture; step 5, constructing a one-step detection kit comprising a reactant placing tube and a syringe arranged in the reactant placing tube, then placing a reaction mixture at the bottom of the reactant placing tube, mixing crRNA and Cas12a protein, and then pre-loading the mixture into the syringe; and step 6, after the reaction mixture reacts, pushing a piston of an injector to enable the crRNA and the Cas12a protein to be mixed with the reaction mixture and react, and then carrying out blue light excitation to obtain a detection result, wherein when the crRNA is designed, a cutting site of the crRNA on a target sequence and a complementary strand of the crRNA is positioned on a primer amplicon sequence, and then according to the designed sequence of the crRNA, chemical synthesis is directly carried out or a crRNA in-vitro transcription vector is constructed and in-vitro transcription and purification are carried out by using a T7 transcription kit.

In the one-step nucleic acid detection method of CRISPR/Cas12a provided by the invention, the method also has the following characteristics: the specific extraction process of the target to be detected in the step 4 comprises the following substeps: step 4-1, taking 1mL of overnight-cultured bacterial liquid of bacteria to be detected, centrifuging at 8000r/min for 5min, and then discarding the supernatant to obtain thalli; and 4-2, washing and coating the thalli twice by using 500ul of physiological saline, then re-suspending the thalli by using a TE buffer solution with the pH value of 7.6, boiling for 10min, immediately freezing for 5min at the temperature of-20 ℃, centrifuging for 5min at the speed of 12000r/min, then taking a supernatant, and taking the supernatant as a target to be detected for later use.

In the one-step nucleic acid detection method of CRISPR/Cas12a provided by the invention, the method also has the following characteristics: step 6-1, placing the reaction mixture in a constant temperature detector at 37 ℃ for real-time signal monitoring, wherein the reaction time is 5-10min, and obtaining the reacted reaction mixture; and 6-2, pushing a piston of the injector to mix the crRNA, the Cas12a protein and the reacted reaction mixture, continuing to react at 37 ℃ for 40min, exciting by using blue light, and observing by naked eyes to obtain a detection result.

Action and Effect of the invention

The one-step nucleic acid detection method of CRISPR/Cas12a, which is related by the invention, has the advantages of rapidness, accuracy, convenience and the like, and is beneficial to popularization in a basic level inspection mechanism. Compared with the existing CRISPR/Cas-based nucleic acid detection technology, the method has the advantages that the reaction time is shortened to 30-50min, and the operation is simpler and more convenient; in addition, when the constructed one-step detection kit is used for detection, RPA amplification and CRISPR detection are combined, the operation is completed in one step, the cover opening operation is avoided, aerosol pollution cannot be generated, and the method is more beneficial to practical application.

Drawings

Fig. 1 is a schematic flow diagram of a CRISPR/Cas12a one-step nucleic acid detection method in an embodiment of the invention;

FIG. 2 shows the result of agarose gel electrophoresis of the product of amplification using different upstream and downstream primers for RPA in the examples of the present invention;

FIG. 3 shows fluorescence detection results of Cas12a/crRNA at different concentrations of additives in the present example;

fig. 4 is a result of detection of fluorescent signals at different Cas12a addition times in an embodiment of the present invention;

FIG. 5 is a schematic view of detection by a one-step detection kit in the example of the present invention.

FIG. 6 shows the results of sensitivity for detecting Listeria monocytogenes in an embodiment of the present invention;

FIG. 7 shows the results of cross-reactions for detecting different strains in examples of the present invention;

FIG. 8 shows the results of 20 Listeria strains tested in the examples of the present invention.

Detailed Description

In order to make the technical means and functions of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the accompanying drawings.

< example >

The reagents, instruments and strains used in this example were as follows:

the main reagents are as follows: LbCas12a bokelais biotechnology ltd, guangzhou;XP Beckman Coulter; the RPA amplification kit Weifang 'an Pu' er future Biotechnology Co., Ltd; primer sequence Biotechnology engineering, Inc.; HiScribe T7 Quick High Yield RNA Synthesis Kit New England Biolabs.

The main apparatus is as follows: microplate reader SpectraMax M2 was purchased from Molecular Devices; nanodrop 2000C was purchased from Thermo Scientific; HiScribe T7 Quick HighYield RNA Synthesis kit New England Biolabs.

The strains used are shown in table 1:

TABLE 1 Strain names and corresponding numbering

Fig. 1 is a schematic flow diagram of a CRISPR/Cas12a one-step nucleic acid detection method in an embodiment of the invention.

As shown in fig. 1, the CRISPR/Cas12a one-step nucleic acid detection method of the present embodiment includes the following steps:

in this example, Listeria monocytogenes Lm-EGD-e was selected for detection.

Step 1, downloading an Lm-EGD-e hly gene sequence on NCBI, designing RPA upstream and downstream primers by using Primer5.0, carrying out specificity screening, amplifying a target sequence by using the RPA upstream and downstream primers subjected to the specificity screening to obtain a primer amplicon sequence, carrying out specificity verification on the primer amplicon sequence by using BLAST, and searching for a 5 '-TTTN-3' sequence in the primer amplicon sequence.

The RPA upstream and downstream primers that can be used in this example are shown in table 2:

TABLE 2 RPA primer sequences

When the specificity of the RPA upstream and downstream primers is screened, the primers in the table 1 are respectively used for amplifying target sequences, and screening is carried out through agarose gel electrophoresis results.

FIG. 2 shows the result of agarose gel electrophoresis of the amplified products using different upstream and downstream primers for RPA in the examples of the present invention.

As shown in FIG. 2, the primers represented by the respective symbols in the figure were M: Marker, 1: RPA-158, 2: RPA-264, 3: RPA-261, and 4: RPA-291, and the specificity and amplification efficiency of the RPA-264-F/R primer were the highest as seen from the results of agarose gel electrophoresis, and thus the amplification was carried out using the selected primers in this example.

Step 2, designing and detecting the required specific crRNA according to the target sequence, in the embodiment, selecting a crRNA sequence by using CRISPR-DT design, and enabling the cleavage site of the crRNA on the target sequence and the complementary strand of the crRNA to be positioned on a primer amplicon sequence, wherein the specific process of synthesizing the crRNA comprises the following steps of forming crRNA-F by using a T7 promoter (TAATACGACTCACTATAGG) + a scaffold sequence (aatttctactaagtgtagat) + a target sequence (20-23 bp after the target DNPAM sequence) of LbCas12a, and carrying out reverse complementation to obtain crRNA-R, wherein the T7 promoter enables the annealed double strand to be recognized and transcribed by T7 RNA polymerase, and the scaffold sequence can be combined with LbCas12 a. The two oligonucleotide chains of the synthesized crRNA-F/R are shown in Table 3, and after annealing, a DNA double strand is obtained, and then in vitro transcription is carried out to obtain the crRNA required by detection.

TABLE 3 CRRNA template primer sequences

And 3, designing a single-stranded DNA (deoxyribonucleic acid) reporter molecule for fluorescent signal detection and blue light excitation visual detection, wherein the single-stranded DNA reporter molecule adopts a 5-base random single-stranded DNA molecule with two ends respectively provided with FAM (fatty acid) and BHQ1 groups, and the single-stranded DNA reporter molecule selected in the embodiment is shown in the following table 4.

TABLE 4 Single-stranded DNA reporter sequences

And 4, mixing the single-stranded DNA reporter molecule, the RPA upstream and downstream primers, the freeze-dried RPA reaction particles, magnesium acetate, the RPA hydration buffer solution and the target to be detected to obtain a reaction mixture.

The specific extraction process of the target to be detected in the step 4 comprises the following substeps:

step 4-1, taking 1mL of overnight-cultured bacterial liquid of bacteria to be detected, centrifuging at 8000r/min for 5min, and then discarding the supernatant to obtain thalli;

and 4-2, washing and coating the thalli twice by using 500ul of physiological saline, then re-suspending the thalli by using a TE buffer solution with the pH value of 7.6, boiling for 10min, immediately freezing for 5min at the temperature of-20 ℃, centrifuging for 5min at the speed of 12000r/min, then taking a supernatant, and taking the supernatant as a target to be detected for later use.

Step 5, constructing a one-step detection kit comprising a reactant placing tube and a syringe placed in the reactant placing tube, then placing the reaction mixture at the bottom of the reactant placing tube, mixing the crRNA and the Cas12a protein, and pre-loading the mixture into the syringe.

In this embodiment, the maximum release of the enzyme activity in the system is ensured by adjusting the concentration of Cas12a/crRNA, and the specific process is as follows: cas12a and crRNA with different concentrations are respectively mixed with the reaction mixture and placed in a constant temperature detector at 37 ℃ for real-time signal monitoring, the reaction time is 60min, fluorescence signals are collected once per second, the detection result is interpreted, Cas12a/crRNA with the optimal mixed concentration is obtained according to the detection result and is mixed and preloaded in an injector, and FIG. 3 shows the fluorescence detection results of Cas12a and crRNA with different addition concentrations in the embodiment of the invention.

As shown in fig. 3, in the graph, (a) is the fluorescence detection result of Cas12a at different addition concentrations, and as shown by the result, the highest fluorescence detection value can be obtained when the addition amount of the target to be detected is 1nM and the addition concentration of Cas12a is 60nM, and in the graph, (b) is the fluorescence detection result of crRNA at different addition concentrations, and as shown by the result, the highest fluorescence detection value can be obtained when the addition amount of the target to be detected is 1nM and the addition concentration of crRNA is 120nM, so that according to the concentration of the target to be detected, when the addition amount of the target to be detected is 1nM, the addition concentration of Cas12a is 60nM and the addition concentration of crRNA is 120nM as the optimum mixed concentration.

In this embodiment, 2ul of target to be detected is directly mixed with crRNA, Cas12a, single-stranded DNA reporter, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer, NEB buffer2.1 and RNase Inhibitor to obtain a mixed solution, the mixed solution is incubated at a constant temperature of 37 ℃ and subjected to fluorescence detection, and the components of the mixed solution are distributed as shown in Table 5 and Table 5

According to the fluorescence detection result, when Cas12a is directly added to the reaction system, the fluorescence signal value is lower, probably because the initial template is reduced due to the target shearing of Cas12a/crRNA, so the Cas12a addition time needs to be optimized, and fig. 4 is the fluorescence signal detection result of different Cas12a addition times in the embodiment of the present invention.

As shown in FIG. 4, higher fluorescence signal values can be obtained by adding Cas12a after the reaction mixture is reacted for 5-10min, so that the crRNA and Cas12a protein are added for reaction after the reaction mixture is reacted for 5-10 min.

And 6, after the reaction mixture reacts, pushing a piston of the injector to mix the crRNA and the Cas12 protein with the reaction mixture for reaction, and exciting by blue light to obtain a detection result.

FIG. 5 is a schematic view of detection by a one-step detection kit in the example of the present invention.

As shown in fig. 5, step 6 includes the following sub-steps:

step 6-1, placing the reaction mixture in a constant temperature detector at 37 ℃ for real-time signal monitoring, wherein the reaction time is 5-10min, and obtaining a reaction mixture after reaction;

and 6-2, pushing a piston of the injector to mix the crRNA, the Cas12a protein and the reacted reaction mixture, continuing to react at 37 ℃ for 40min, exciting by using blue light, and observing by naked eyes to obtain a detection result.

In this embodiment, the detection sensitivity of the present invention is also tested, and the specific process is as follows:

selecting Lm-EGD-e single colony in culture medium, shake culturing at 37 deg.C for 12 hr, and counting to obtain pure bacteria solution with concentration of 5.1 × 10⁹cfu/mL, diluting the cultured bacterial liquid to 10 with normal saline⁸、10⁶、10⁴、10²、10¹、10⁰CFU/mL, extracting the target to be detected from the bacterial liquid with each concentration according to the step 4, taking 2ul of the target to be detected from each concentration during the test, adding the target to be detected into the detection system, using water negative control, and repeating the steps for three times.

FIG. 6 shows the results of sensitivity for detecting Listeria monocytogenes in an embodiment of the present invention.

As shown in FIG. 6, the concentration of 10 was added⁹、10⁷、10⁵、10²、10¹、10⁰CFU/mL target to be detected, the fluorescence signal is obviously higher than that of the negative control group, 10 drops are added⁰The difference between the fluorescence signal of the CFU/mL target to be detected and the negative control is not obvious, which indicates that the sensitivity of the CRISPR/Cas12a one-step nucleic acid detection method reaches 10¹CFU/mL, three replicates were 10¹CFU/mL, therefore, the cloud has high detection sensitivity and good stability, and the minimum detection concentration is 10¹CFU/mL。

In this example, the detection specificity of the present invention was also tested, and the specific process was as follows:

detection of 15 non-listeria monocytogenes strains by using pre-mixed CRISPR/Cas12a for detecting listeria monocytogenes, and culturing the strains to 10⁶CFU/mL of bacteria and observed results.

FIG. 7 shows the results of cross-reactions for detecting different strains in the examples of the present invention.

As shown in FIG. 7, the bacteria represented by the respective reference numerals are as follows: 1: listeria monocytogenes EGD-e, 2: enterobacter sakazakii ATCC29004, 3, Shigella flexneri ATCC12022, 4: shigella boydii ATCC9207, 5: shigella sonnei CMCC51592, 6: salmonella typhimurium ATCC14028, 7: salmonella enteritidis ATCC13076, 8: salmonella choleraesuis CMCC50018, 9: salmonella paratyphi type a CMCC50093, 10: salmonella paratyphi b CMCC50004, 11: salmonella paratyphi type c CMCC50118, 12: staphylococcus aureus ATCC 27660, 13: enterobacter sakazakii ATCC29004, 14: shigella flexneri ATCC12022, 15: shigella boydii ATCC9207, 16: shigella sonnei CMCC51592,

according to the detection result, the CRISPR/Cas12a one-step nucleic acid detection method can specifically detect the Listeria monocytogenes, and has good specificity.

In this example, 20 listeria strains nos. 1-20 in table 1 were tested and verified by comparison using nucleic acid detection gold standard qPCR.

FIG. 8 shows the results of 20 Listeria strains tested in the examples of the present invention.

As shown in fig. 8, the left side shows qPCR results, and the right side shows the detection results of the present invention, wherein 20 strains detected in the figure correspond to the following: the A lattices respectively from left to right are as follows: listeria griffithii CICC21670, Listeria ovis CICC21663, Listeria griffithii Lgr0001, Listeria williamsii CICC21672, Listeria lnokhei CICC10297,

the B lattices from left to right are respectively: listeria monocytogenes AB2483, Listeria monocytogenes Lm-F204, Listeria monocytogenes CMCC54003, Listeria monocytogenes CMCC54007, Listeria monocytogenes NCTC5348,

the C lattices from left to right are respectively: listeria monocytogenes ATCC19112, Listeria monocytogenes ATCC19115, Listeria monocytogenes ATCC19116, Listeria monocytogenes ATCC19117, Listeria monocytogenes ATCC19118,

the D lattices from left to right are respectively: listeria monocytogenes EGD-e, Listeria monocytogenes BAA751, Listeria monocytogenes 10403S, Listeria monocytogenes ScottA, Listeria monocytogenes F2365,

according to the detection result, the detection result is completely consistent with the qPCR result, and the detection can be rapidly and accurately finished.

Effects and effects of the embodiments

The one-step nucleic acid detection method for the CRISPR/Cas12a has the advantages of being fast, accurate, convenient and the like, and is favorable for popularization in basic level inspection mechanisms. Compared with the existing CRISPR/Cas-based nucleic acid detection technology, the reaction time of the embodiment is shortened to 30-50min, and the operation is simpler and more convenient; in addition, when the constructed one-step detection kit is used for detection, RPA amplification and CRISPR detection are combined, the operation is completed in one step, the cover opening operation is avoided, aerosol pollution cannot be generated, and the one-step detection kit is more beneficial to practical application.

The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.