Detection system for visually detecting porcine Japanese encephalitis virus based on RT-RAA-CRISPR / Cas12a system and application

Abstract

The invention discloses a detection system for visually detecting a swine Japanese encephalitis virus based on an RT-RAA-CRISPR / Cas12a system, and the visual detection of the JEV is realized by designing crRNA and RAA amplification primers for a JEV NS1 gene conserved region and triggering the breakage of a double-labeled ssDNA reporter molecule by using the specific recognition and trans-cleavage activity of Cas12a protein to an RAA amplification product. The lower detection limit of the method is 2.04 * 10 < 0 > copies / [mu] L; the clinical sample detection rate is 61.0% and is equivalent to that of 63.4% of PCR (Polymerase Chain Reaction). Therefore, the invention establishes a nucleic acid diagnosis method with strong specificity, high sensitivity, short detection time and simple operation, and the method is also suitable for pig farm on-site epidemic situation screening, animal quarantine ports, grassroots veterinary stations, remote area public health monitoring and other aspects.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a detection system and application of porcine Japanese encephalitis virus based on the RT-RAA-CRISPR / Cas12a system for visual detection. Background Art

[0002] Japanese Encephalitis Virus (JEV) is a Flaviviridae pathogen transmitted by Culex tritaeniorhynchus mosquitoes, causing zoonotic Japanese encephalitis (JE). Pigs, as the primary reservoir and transmission host of JEV, occupy a central position in the virus transmission chain. JEV infection can cause reproductive disorders such as abortion and stillbirth in sows and orchitis in boars, causing significant economic losses to the pig industry. JEV is particularly prevalent in Asia. For example, epidemiological surveys in Yunnan, Shaanxi, and Dongting Lake in my country have shown that the infection rate in pig herds is as high as 18.5%, and the mosquito carriage rate can reach 42.9%. In addition, the virus has significant genotypic variation (such as the coexistence of genotypes I and III), further increasing the difficulty of prevention and control.

[0003] The detection technology of porcine Japanese encephalitis virus (JEV) currently relies mainly on traditional molecular biology and immunological methods, but they have significant limitations in sensitivity, ease of operation and field applicability. Li et al. designed a one-step RT-PCR method based on the primers of the JEV E gene, with a sensitivity of 10pg total RNA, but it relies on a thermal cycler and professional operators, and the detection time takes 2-3 hours. Du Juan et al. achieved rapid detection (sensitivity 10 3 TCID 50 / 0.2mL), but it requires the design of complex primers (3 pairs of primers) and relies on SYBR GreenⅠ fluorescent dye for result interpretation, which cannot be visualized with the naked eye. In the article "Establishment of RPA-CRISPR / Cas12a Detection Method Based on NS3 Gene of Japanese Encephalitis Virus", a detection method with simple operation (rapid detection of JEV within 30 minutes) and good sensitivity was established using RPA-CRISPR / Cas12a detection method, which has low requirements for instruments and equipment (the detection results can be judged by the naked eye under blue light excitation or directly through the test strip). The detection limit of the entire test reaction reached 10 2 The reproducibility of the assays was good, with a mean of 100 copies / μL. Repeatability tests showed good inter- and intra-batch reproducibility. However, the sensitivity of JEV detection still needs to be further improved. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide a detection system for visual detection of porcine Japanese encephalitis virus based on the RT-RAA-CRISPR / Cas12a system.

[0005] The second object of the present invention is to provide an application of the above detection system in the preparation of a product for detecting porcine Japanese encephalitis virus.

[0006] The third object of the present invention is to provide a kit comprising the above detection system.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a detection system for visually detecting porcine Japanese encephalitis virus based on an RT-RAA-CRISPR / Cas12a system. The detection system comprises a RAA amplification system, a CRISPR / Cas12a detection system, and an ssDNA probe; the RAA amplification system comprises RAA primers, and the nucleotide sequences of the RAA primers are respectively shown in SEQ ID NOs. 12 to 13; the CRISPR / Cas12a detection system comprises a Cas12a protein and crRNA, and the nucleotide sequences of the crRNA are respectively shown in SEQ ID NO. 7.

[0009] RT-RAA (recombinase-mediated amplification) is an isothermal nucleic acid amplification technology that combines the characteristics of recombinase polymerase amplification (RPA) and reverse transcriptase amplification (RT). It uses recombinases and primers obtained from bacteria or fungi to quickly amplify target fragments at 37°C. RAA uses specific primers under the action of reverse transcriptase to reverse transcribe the organism's mRNA into cDNA as a template, and then uses the reverse transcribed cDNA as a template for DNA amplification. Because reverse transcriptase is used in RAA technology, it is particularly suitable for amplification detection targeting RNA. The RT-RAA-CRISPR / Cas12a system is a visual detection technology that combines reverse transcriptase-mediated nucleic acid amplification (RT-RAA) with CRISPR / Cas12a gene editing tools. Its core principle is based on the cascade reaction of nucleic acid amplification-target recognition-signal release.

[0010] The present invention is directed to the conserved region of the porcine Japanese encephalitis virus JEV NS1 gene, and designs three groups of one-to-one corresponding crRNA-primer combinations (upstream primers with T7 transcript sequence and crRNA backbone sequence required for Cas12a recognition, and downstream primers with crRNA backbone sequence and target gene sequence), and amplifies to obtain crRNA; one-to-one corresponding RAA amplification primers are designed for each crRNA target, and crRNA screening is performed respectively, and JEV-primer3 is selected for subsequent experiments; Cas12a protease concentration, Cas12a protease: crRNA ratio, ssDNA probe concentration and optimum reaction temperature are further optimized to establish an optimal RT-RAA-Cas12a-JEV detection system, thereby establishing a RT-RAA-Crispr-Cas12a-JEV nucleic acid diagnostic method with short detection time, strong specificity and high sensitivity. The system can trigger the breakage of double-labeled ssDNA reporter molecules through the specific recognition and trans-cleavage activity of Cas12a protein to RAA amplified products, thereby achieving visual detection of JEV. It is suitable for on-site epidemic screening at pig farms, animal quarantine ports, grassroots veterinary stations, and public health monitoring in remote areas. Specifically, the sample to be tested is first amplified by RT-RAA. Then, under the mediation of the crRNA sequence, the CRISPR / Cas12a system is guided to recognize and bind to the RAA amplification product and cleave the target double-stranded DNA to activate the non-specific nuclease function. The double-labeled ssDNA reporter molecule in the system is then randomly cleaved to obtain cleavage products, and finally the cleavage products are detected by colorimetric detection for judgment.

[0011] Furthermore, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.

[0012] Furthermore, the RAA amplification system also includes a sample to be tested, a buffer, nuclease-free water, and a magnesium acetate solution.

[0013] Furthermore, the concentration of the RAA primer in the RAA amplification system is 10 μM to 20 μM; the concentration of the magnesium acetate solution is 10 to 17.5 mM.

[0014] Preferably, the concentration of the RAA primer in the RAA amplification system is 10 μM; the concentration of the magnesium acetate solution is 14 nM.

[0015] Furthermore, the CRISPR / Cas12a detection system also includes Cas12a protein, crRNA, buffer, and nuclease-free water.

[0016] Furthermore, the Cas12a protein is LbCas12a.

[0017] Furthermore, the concentration ratio of the Cas12a protein to crRNA is 1:1-5.

[0018] Furthermore, the final concentration of the Cas12a protein is 100-1000 nM.

[0019] Furthermore, the final concentration of the ssDNA probe is 500-1250 nM.

[0020] Preferably, the final concentration of Cas12a protein in the CRISPR detection system is 800 nM; the final concentration ratio of Cas12a protein and crRNA is 1:4; and the final concentration of ssDNA probe is 1000 nM.

[0021] Furthermore, the detection system also includes porcine Japanese encephalitis virus standard samples of different concentrations, which can be used for on-site visual quantitative comparison of porcine Japanese encephalitis virus.

[0022] The present invention designs crRNA and RAA amplification primers for the conserved region of the JEV NS1 gene, utilizes the specific recognition and trans-cleavage activity of the Cas12a protein on the RAA amplification product, triggers the breakage of the double-labeled ssDNA reporter molecule (FAM probe), and realizes the visual detection of JEV. The sensitivity of this nucleic acid diagnostic method is consistent with the national standard PCR detection method, with a detection limit of 2.04×10^0 copies / μL; the detection rate of clinical samples is 61.0% (25 / 41), which is equivalent to 63.4% (26 / 41) of PCR. This method is suitable for on-site epidemic screening in pig farms, animal quarantine ports, grassroots veterinary stations, and public health monitoring in remote areas.

[0023] Therefore, the present invention provides the use of the detection system in preparing a product for detecting porcine Japanese encephalitis virus.

[0024] The present invention also provides a kit comprising the above detection system.

[0025] Furthermore, the method for using the kit comprises the following steps:

[0026] S1. Place the RAA amplification system at the bottom of a centrifuge tube, incubate at 36-42°C for 8-12 minutes, and then add the CRISPR / Cas12a detection system and ssDNA probe. Alternatively, place the RAA amplification system at the bottom of a centrifuge tube, place the CRISPR / Cas12a detection system and ssDNA probe on the cap of the centrifuge tube, incubate at 36-42°C for 8-12 minutes, and then centrifuge briefly.

[0027] S2. Place the centrifuge tube from step S1 in a fluorescent quantitative PCR detection system for reaction or in a water bath for reaction.

[0028] Furthermore, step S2 is to place the centrifuge tube of step S1 in a fluorescent quantitative PCR detection system for reaction at a reaction temperature of 37-42°C, and perform a reading every 5 minutes, or place the tube in a water bath, react at 37-42°C for 10-30 minutes, and read the results using a blue light gel cutting instrument.

[0029] Furthermore, the reaction temperature is 42°C.

[0030] This method uses a single-tube reaction system to integrate retroviral nucleic acid, RAA amplification, and CRISPR detection steps, simplifying the Japanese encephalitis virus (JE) assay workflow to a total time of 40 minutes or less, reducing the risk of aerosol contamination. Test results are visualized through fluorescence signals. The entire reaction is performed at 37-42°C, eliminating the need for specialized thermostats. The simple operation makes it suitable for grassroots laboratories or field screening.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a detection system and application for visual detection of porcine Japanese encephalitis virus based on the RT-RAA-CRISPR / Cas12a system. The present invention designs crRNA and RAA amplification primers for the conserved region of the JEV NS1 gene, utilizes the specific recognition and trans-cleavage activity of the Cas12a protein on the RAA amplification product, triggers the breakage of the double-labeled ssDNA reporter molecule (FAM probe), and realizes visual detection of JEV. The detection limit of this method is 2.04×10^0 copies / μL; the detection rate of clinical samples is 61.0% (25 / 41), which is equivalent to 63.4% (26 / 41) of PCR. Therefore, the present invention establishes a RT-RAA-Crispr-Cas12a-JEV nucleic acid diagnostic method with strong specificity, high sensitivity, short detection time and simple operation. The method is also suitable for on-site epidemic screening in pig farms, animal quarantine ports, grassroots veterinary stations and public health monitoring in remote areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Screening for suitable sequences in the JEV conserved regions.

[0034] Figure 2 For the screening of crRNA-primers. Figure 2 a shows the change of fluorescence intensity of JEV-crRNA-primer1~3 over time; b shows the fluorescence intensity of JEV-crRNA-primer1~3 after 30 minutes of reaction; c shows the result displayed by blue light gel cutting instrument.

[0035] Figure 3 To explore the optimal Cas12a concentration. Figure 3a shows the change of fluorescence intensity of Cas12a with different concentrations over time; b shows the fluorescence intensity of Cas12a with different concentrations after 30 minutes of reaction; c shows the result displayed by the blue light gel cutting instrument.

[0036] Figure 4 To explore the optimal Cas12a:crRNA ratio. Figure 4 a shows the change of fluorescence intensity of Cas12a:crRNA with different ratios over time; b shows the fluorescence intensity of Cas12a:crRNA reaction for 30 minutes with different ratios; c shows the result displayed by blue light gel cutting instrument.

[0037] Figure 5 To explore the optimal concentration of FAM probe (dual-labeled ssDNA reporter molecule). Figure 5 Figure a shows the change of FAM fluorescence intensity with time at different concentrations; b shows the fluorescence intensity of FAM at different concentrations after 30 minutes of reaction; and c shows the result displayed by the blue light gel cutting instrument.

[0038] Figure 6 To explore the optimal temperature for the reaction. Figure 6 a shows the change of fluorescence intensity over time at different temperatures; b shows the fluorescence intensity after 30 minutes of reaction at different temperatures; c shows the result displayed by the blue light gel cutting instrument.

[0039] Figure 7 This is to verify the specificity of the RT-RAA-Cas12a-JEV reaction system.

[0040] Figure 8 This is a sensitivity verification of the RT-RAA-Cas12a-JEV reaction system.

[0041] Figure 9 Detect clinical samples for the RT-RAA-Cas12a-JEV reaction system. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] 1. Experimental Materials

[0045] Table 1 Main biological materials

[0046]

[0047] Table 2 Main reagents

[0048]

[0049] Example 1 Establishment of a system for visual detection of porcine Japanese encephalitis virus (JEV) based on the RT-RAA-CRISPR / Cas12a system

[0050] 1. Experimental Methods

[0051] 1. Expression and purification of Cas12a protein

[0052] The pET28a-LbCas12a expression strain was revived and induced for expression. After purification, it was stored at -80 degrees Celsius for later use.

[0053] 2. Preparation of crRNA

[0054] The full-length JEV reference strains included in GenBank were selected and downloaded, and multiple sequence alignment was performed using the Align method in MEGA X software to screen for suitable sequences in the conserved regions, such as Figure 1 As shown in Table 3, crRNA was designed. An upstream primer with a T7 transcript sequence and the crRNA backbone sequence required for Cas12a recognition was designed, and a downstream primer with a crRNA backbone sequence and a target gene sequence was designed, as shown in Table 3.

[0055] Table 3 JEV crRNA primers

[0056]

[0057]

[0058] Annealing was performed using a PCR instrument: 95°C for 2 minutes to fully denature the oligo. Starting at 95°C, the temperature was decreased by 0.1°C every 8 seconds until it reached 25°C. T7-12a-F and the downstream components were fused into double-stranded DNA. The annealing reaction system is shown in Table 4. An equal volume of Taq enzyme (2× Rapid Taq Master Mix) was placed in a metal bath at 95°C for 2 minutes to activate the Taq enzyme. The bath was then adjusted to 72°C, and the annealed product was added. The mixture was incubated for 10 minutes to fully extend the combined duplexes into complete double strands.

[0059] Table 4 Annealing reaction system

[0060]

[0061] The obtained extension reaction products were purified using OMEGA Cycle Pure Kit D6492. The reaction system was configured according to the instructions of T7 High Yield RNA Transcription Kit, and the reaction was carried out at 37°C overnight. The double-stranded DNA was transcribed in vitro to obtain crRNA, as shown in Table 5. The harvested RNA was used Purify with RNA Clean Up Kit, determine the concentration and store at -80℃.

[0062] Table 5 JEV crRNA sequence

[0063]

[0064] 3. RAA primer design

[0065] One-to-one corresponding RAA amplification primers were designed for each crRNA target site, and the primer sequences are shown in Table 6.

[0066] Table 6 RAA amplification primers

[0067]

[0068] 4. JEV nucleic acid extraction

[0069] JEV nucleic acid was extracted using Omega Viral RNA Kit R6874. For specific steps, see the product manual.

[0070] 5. Establishment of RT-RAA-Cas12a-JEV reaction system

[0071] In the clean room, take out the enzyme-free centrifuge tube, add the RT-RAA system (RT-RAA nucleic acid amplification kit) for removing viral nucleic acid to the bottom of the centrifuge tube (Table 7), and add the Cripr-Cas12a cutting system to the tube cap (Table 8). The viral nucleic acid and 0.5 μL of magnesium acetate solution were added to the amplification system at the bottom of the centrifuge tube in sequence, incubated at 37 ° C for 10 minutes, and centrifuged instantaneously. The centrifuge tube was immediately placed in a fluorescent quantitative PCR detection system for reaction. The reaction temperature was 37 degrees Celsius and readings were taken every 5 minutes. At the same time, the same reaction system was placed in a water bath, reacted at 37 ° C for 30 minutes, and the results were read using a blue light gel cutting instrument.

[0072] Table 7 RT-RAA reaction system

[0073]

[0074]

[0075] Table 8 Crispr-Cas12a cleavage reaction system

[0076]

[0077] 6. Optimization of RT-RAA-Cas12a-JEV reaction system

[0078] The crRNA screening, Cas12a protease concentration optimization, Cas12a protease: crRNA ratio optimization, ssDNA probe concentration optimization and optimal reaction temperature exploration were carried out respectively. When using the instrument to read the fluorescence value, three repeated experiments were set for each system.

[0079] (1) crRNA screening

[0080] Three crRNAs, JEV-crRNA1, JEV-crRNA2, and JEV-crRNA3, were designed targeting the conserved regions of the JEV NS1 gene. RAA amplification primers were then designed for each crRNA recognition site. The reaction system was configured and performed using a fluorescent quantitative PCR system at 37°C, with readings taken every 5 minutes. The same system was then incubated in a water bath at 37°C for 30 minutes, and the results were verified using a blue light gel exciter.

[0081] like Figure 2 As shown in the figure, the order of fluorescence intensity from strong to weak is JEV-crRNA-primer3>JEV-crRNA-primer2>JEV-crRNA-primer1. Therefore, JEV-crRNA-primer3 was selected for subsequent experiments.

[0082] (2) Optimization of Cas12a protease concentration

[0083] The reaction system was configured so that the final concentrations of LbCas12a were 0 nM (negative control), 100 nM, 250 nM, 500 nM, and 800 nM, respectively. Subsequently, the reaction was carried out in a fluorescent quantitative PCR detection system at a reaction temperature of 37 ° C and a reading was taken every 5 minutes. In addition, the same system was reacted in a water bath at 37 ° C for 30 minutes, and the results were checked using a blue light gel cutting instrument.

[0084] like Figure 3 As shown, when the final concentration of Cas12a is 800nM, a higher reaction efficiency can be obtained.

[0085] (3) Optimization of Cas12a protease:crRNA ratio

[0086] The final concentration of Cas12a nuclease was 800 nM, and the Cas12a:crRNA ratios were adjusted to 1:1, 1:2, 1:3, and 1:4, respectively. Reactions were performed using a fluorescent quantitative PCR system at 37°C, with readings taken every 5 minutes. The same system was then incubated in a water bath at 37°C for 30 minutes, and the results were verified using a blue light gel exciter.

[0087] like Figure 4 As shown in the figure, when Cas12a:crRNA=1:4, a faster reaction rate can be obtained, so subsequent experiments make Cas12a:crRNA=1:4.

[0088] (4) Optimization of ssDNA probe concentration

[0089] The final probe concentrations in the reaction system were 0 nM (negative control), 500 nM, 750 nM, 1000 nM, and 1250 nM, respectively. Reactions were performed in a fluorescent quantitative PCR detection system at 37°C, with readings taken every 5 minutes. The same system was also incubated in a water bath at 37°C for 30 minutes, and the results were verified using a blue light gel exciter.

[0090] like Figure 5 As shown in the figure, the reaction rate increases with increasing probe concentration, reaching a peak when the probe concentration reaches 1000 nM. Further increases in probe concentration subsequently decrease the reaction efficiency. Therefore, the final probe concentration in subsequent experiments was 1000 nM.

[0091] (5) Optimization of optimal reaction temperature and time

[0092] The reaction system was placed in a fluorescent quantitative PCR detection system and reacted at 30, 37.6, and 42 degrees Celsius, with readings taken every 5 minutes. Additionally, the same system was reacted in a water bath at 30, 37.6, and 42 degrees Celsius for 30 minutes, and the results were verified using a blue light gel exciter.

[0093] like Figure 6 As shown, obvious fluorescence signals can be detected or seen at 30, 37.6, and 42 degrees Celsius after 10 minutes of reaction; when the reaction temperature is 42 degrees Celsius, the reaction efficiency is significantly higher than that at 30 degrees Celsius or 37.6 degrees Celsius.

[0094] Therefore, the present invention selects the concentration of JEV-crRNA-primer3 and Cas12a protein as 800 nM; the concentration ratio of Cas12a protein and crRNA is 1:4; the concentration of ssDNA probe is 1000 nM; and the reaction temperature is 42 degrees Celsius as the optimal reaction system for subsequent RT-RAA-Cas12a-JEV nucleic acid detection method specificity and sensitivity detection.

[0095] Example 2 Validation of RT-RAA-Cas12a-JEV Nucleic Acid Detection Method

[0096] 1. Experimental Methods

[0097] 1. Specificity detection

[0098] The nucleic acids of common clinical swine pathogens JEV, CSFV, ASFV, PRRSV, PRV, PCV2, and PPV were tested, and a negative control group was set up. Each sample was repeated three times and the results were recorded.

[0099] 2. Sensitivity test

[0100] The JEV nucleic acid template was gradient diluted to 2.04×10^4~2.04×10^0copies / μL and detected using RT-PCR (GB / T 18638-2021) and RT-RAA-Cas12a-JEV detection methods, respectively. Each gradient was set up with three repeated experiments to compare the sensitivity of various methods.

[0101] 2. Experimental Results

[0102] 1. The results are as follows Figure 7 As shown in the figure, only the reaction with JEV nucleic acid as template was positive. No specific fluorescent signal was observed for CSFV, ASFV, PRRSV, PRV, PCV2, PPV, or the negative control, indicating that the method has good specificity.

[0103] 2. When the RT-RAA-Cas12a-JEV method is used for detection, the reaction system is tested with a fluorescent quantitative PCR detection system. The reaction temperature is 42 degrees Celsius and a reading is taken every 5 minutes. At the same time, the same system is reacted in a water bath at 42 degrees Celsius for 30 minutes, and the results are checked using a blue light gel cutting instrument. The results are as follows: Figure 8 As shown in the figure, the detection limit of RT-RAA-Cas12a-JEV is 2.04×10^0 copies / μL, and its sensitivity is consistent with that of RT-PCR.

[0104] Example 3 Clinical sample detection

[0105] 41 clinical nucleic acid samples were tested using RT-PCR (GB / T 18638-2021) and RT-RAA-Cas12a-JEV methods to evaluate the clinical applicability of the detection method established in this study.

[0106] like Figure 9 As shown, 26 JEV positive samples were detected by RT-PCR, with a detection rate of 63.4% (26 / 41); 25 JEV positive samples were detected by RT-RAA-Cas12a-JEV, with a detection rate of 61.0% (25 / 41).

Claims

1. A detection system for visual detection of porcine Japanese encephalitis virus based on the RT-RAA-CRISPR / Cas12a system, characterized in that: The detection system includes a RAA amplification system, a CRISPR / Cas12a detection system and an ssDNA probe; the RAA amplification system contains RAA primers, and the nucleotide sequences of the RAA primers are respectively shown in SEQ ID NOs. 12 to 13; the CRISPR / Cas12a detection system contains Cas12a protein and crRNA, and the nucleotide sequences of the crRNA are respectively shown in SEQ ID NO.

7.

2. The detection system according to claim 1, characterized in that The sequence of the ssDNA probe is 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.

3. The detection system according to claim 1, characterized in that The Cas12a protein is LbCas12a.

4. The detection system according to claim 1, characterized in that The concentration ratio of the Cas12a protein to crRNA is 1:1-5.

5. The detection system according to claim 1, characterized in that The final concentration of the Cas12a protein is 100-1000 nM.

6. The detection system according to claim 1, characterized in that The final concentration of the ssDNA probe is 500-1250 nM.

7. The detection system according to claim 1, characterized in that The detection system also includes porcine Japanese encephalitis virus standard samples of different concentrations.

8. Use of the detection system according to any one of claims 1 to 7 in the preparation of a product for detecting porcine Japanese encephalitis virus.

9. A kit, characterized in that The kit comprises the detection system according to any one of claims 1 to 7.

10. The kit according to claim 9, characterized in that The method for using the kit comprises the following steps: S1. Place the RAA amplification system at the bottom of a centrifuge tube, incubate at 36-42°C for 8-12 minutes, and then add the CRISPR / Cas12a detection system and ssDNA probe. Alternatively, place the RAA amplification system at the bottom of a centrifuge tube, place the CRISPR / Cas12a detection system and ssDNA probe on the cap of the centrifuge tube, incubate at 36-42°C for 8-12 minutes, and then centrifuge briefly. S2. Place the centrifuge tube from step S1 in a fluorescent quantitative PCR detection system for reaction or in a water bath for reaction.

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