Primer group for detecting cordate telosma mosaic virus based on RT-RAA and CRISPR / Cas12a and application of primer group
By using the combination of RT-RAA and CRISPR/Cas12a technology in the detection of Yelaixiang mosaic virus, the existing detection methods have been solved, and the rapid detection with high specificity, high sensitivity and low cost are achieved, which is suitable for disease screening of passionflower in the field.
Patent Information
- Application Number
- CN202510225247.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-06-13
AI Technical Summary
Existing methods for detecting night-length mosaic viruses such as serology methods and RT-PCR have problems such as low sensitivity, complex operation, time-consuming and high cost, and it is difficult to meet the needs of rapid detection in the field.
The detection primer set based on RT-RAA and CRISPR/Cas12a, including RT-RAA amplification primer pair and CRISPR/Cas12a detection primer, combined with the rapid RT-RAA reaction and the trans cleavage activity of CRISPR/Cas12a, is used to achieve rapid detection of nycens mosaic virus.
It has achieved high specificity, high sensitivity and low cost Mosaic virus detection, which is suitable for rapid screening and seedling quarantine of passionflower and other crops in the field, and provides technical support for healthy seedling detection and field screening of diseases.
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Figure CN120138221A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a primer set for detecting telosma mosaic virus based on RT-RAA and CRISPR / Cas12a and its application. Background Art
[0002] Passiflora edulis, also known as passion fruit, is a perennial herbaceous vine of the genus Passiflora in the family Passifloraceae. As a tropical fruit with important economic value, it is currently widely planted in tropical and subtropical regions such as Guangdong, Guangxi, Fujian, Hainan, Yunnan, Guizhou, and Taiwan in China. Since Passiflora edulis (passion fruit) is propagated asexually and can be planted year after year, coupled with frequent transportation of seedlings, virus diseases have spread rapidly, seriously affecting the yield and quality of Passiflora edulis and restricting the development of the Passiflora edulis industry. Currently, up to 38 viruses have been reported to infect Passiflora edulis (passion fruit) in the world. Among them, after being infected by telosma mosaic virus (TelMV), the leaves of Passiflora edulis will show mosaic symptoms, that is, the leaves will show yellowish-white mottled mosaic, the leaves will be wrinkled and deformed, and the plants may show dwarfing, weakness and other phenomena, seriously affecting the normal growth and development of plants, resulting in lignification of the pericarp, and seriously affecting the yield and quality of agricultural products. Therefore, it is particularly important to establish a simple, specific, sensitive and rapid detection method.
[0003] Currently, the detection methods for TelMV are mainly serological methods based on virus protein antibodies and reverse transcription-polymerase chain reaction (RT-PCR). The serological methods are mainly enzyme-linked immunosorbent assay (ELISA) and colloidal gold test strips, etc., which are suitable for the detection of a large number of samples, but the sensitivity is lower than that of nucleic acid detection methods. RT-PCR has a high dependence on operators and laboratory instruments and equipment. It requires professional operation to extract total RNA and the PCR instrument needs to perform steps such as denaturation, annealing, and extension. The whole process takes a long time, has a high cost, does not have the advantage of rapid and portable detection, and cannot meet the key conditions for on-site rapid detection.
[0004] Recombinase-aided amplification (RAA) can rapidly amplify target fragments under a constant temperature condition of about 37°C without the need for precise temperature control equipment. Compared with PCR and RT-PCR, the RAA reaction is faster, more sensitive, and has strong specificity. Currently, it has been used for the detection of novel coronavirus, African swine fever virus, avian influenza virus, etc., but there are relatively few reports on its use for the detection of plant viruses.
[0005] In recent years, the gene editing tool CRISPR / Cas system has been increasingly used in the field of nucleic acid detection. Cas12a protein belongs to the 2nd type V CRISPR system effector protein and is an RNA-guided nuclease. CRISPR / Cas12a can recognize a specific protospacer adjacent motif (PAM) sequence of several nucleotides (LbCas12a can recognize 5'-TTTN-3'); under the guidance of crRNA, it cuts double-stranded target DNA (double-strandedDNA, dsDNA); after cutting dsDNA, it can activate its activity of cutting single-stranded DNA (ssDNA). After cutting the target DNA, the Cas12a protein can also cut nearby non-target single-stranded DNA. This cutting activity on non-specific targets is called trans-cutting. A single-stranded DNA reporter gene with a fluorescent group and a fluorescent quenching group at both ends is added to the system. When the target sequence exists in the system, the trans-cleavage activity of the Cas12a protein is activated, the single-stranded DNA reporter gene is chopped up, and the system emits fluorescence to complete the detection process. On the contrary, when the target sequence does not exist, the trans-cleavage activity of the Cas12a protein will not be activated, that is, the system does not fluoresce.
[0006] At present, research teams at home and abroad only use conventional detection methods to detect passion fruit virus diseases, and there has been no report on rapid detection methods for TelMV based on RT-RAA and CRISPR / Cas12a. Summary of the invention
[0007] In view of this, the present invention provides a primer set, a detection method, a kit and applications of tuberose mosaic virus detection based on RT-RAA and CRISPR / Cas12a.
[0008] The primer set for detecting tuberose mosaic virus based on RT-RAA and CRISPR / Cas12a provided by the present invention comprises an RT-RAA amplification primer pair and a CRISPR / Cas12a detection primer;
[0009] The RT-RAA amplification primer pair includes RAA-TelMV-1F and RAA-TelMV-1R, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively;
[0010] The CRISPR / Cas12a detection primer is TelMV-CrRNA, and its nucleotide sequence is shown in SEQ ID NO.3.
[0011] The present invention also provides the use of the above primer set in detecting the virus of Telosma cordata mosaic virus.
[0012] The present invention also provides a reagent or kit containing the above detection primer set; further, the kit further includes a single LbCas12a protein required for the CRISPR / Cas12a detection system, and an ssDNA reporter molecule (FAM-CCCCCCCC-BHQ1) for CRISPR / Cas12a detection, where FAM is 5-carboxyfluorescein and BHQ1 is a fluorescence quencher.
[0013] The present invention also provides a detection method for detecting the virus of Telosma cordata mosaic virus using the above kit, including the following steps: using the nucleic acid of the sample to be tested as a template and detecting it using the above kit;
[0014] Further, the detection method includes the following steps:
[0015] S1, take the sample to be tested, add the crude extraction buffer and steel beads for grinding to obtain a crude extract; wherein, the formula of the crude extraction buffer is: 6% PEG200, 20 mM NaOH;
[0016] S2, perform RT-RAA reaction using the RT-Basic Nucleic Acid Amplification Kit (RAA method) of Hangzhou Zhongce Biotechnology Co., Ltd., take 1 μL of the crude extract and add it to the RT-RAA reaction system. The RT-RAA reaction system is: 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (primer concentration: 10 μmol / L), 1 μL of RAA-TelMV-1R (primer concentration: 10 μmol / L), and make up to 25 μL with ddH 2 O; water bath at 37°C for 20 min;
[0017] S3, take 2 μL of the RT-RAA reaction product and add it to the CRISPR / Cas12a detection system, water bath at 37°C for 10 min. After the reaction is completed, observe the fluorescence of the reaction product under blue light. Fluorescence indicates positive, and no fluorescence indicates negative; wherein, the CRISPR / Cas12a detection system is: 0.4 μL of LbCas12a (10 μmol / L), 0.4 μL of EAPV-crRNA (10 μmol / L), 2 μL of ssDNA (10 μmol / L), 2 μL of 10x Enhanced Buffer, 2 μL of the RT-RAA reaction product, and make up with ddH 2Make up to 20 μL; among them, the formula of 10x Enhanced Buffer is: 100 mM Tris-HCl (pH 9.0), 100 mM NaCl, 150 mM MgCl 2 , 10 mM DTT, 5% PEG-200.
[0018] Compared with the prior art, the present invention has the following beneficial technical effects:
[0019] The present invention designs specific RT-RAA primers and CrRNA through all the published genomic sequences of the Telosma mosaic virus, and establishes a field visual rapid detection method for the Telosma mosaic virus based on RT-RAA and CRISPR / Cas12a, which has the characteristics of high specificity, high sensitivity and low cost. It is widely applicable to the large-scale screening of the Telosma mosaic virus infection in passion fruit (Passiflora edulis) and other crops in the field and the quarantine detection before the transportation of seedlings, providing technical support for the detection of healthy seedlings and the field screening of diseases. Brief Description of the Drawings
[0020] The drawings included in the specification and constituting a part of the specification, together with the specification, illustrate the exemplary embodiments, features and aspects of the present invention, and are used to explain the principles of the present invention.
[0021] Figure 1 For the screening of RT-RAA reaction primers
[0022] Figure 2 For the screening results of RT-RAA reaction primer concentrations;
[0023] Figure 3 For the screening results of RT-RAA reaction temperatures;
[0024] Figure 4 For the screening results of RT-RAA reaction times;
[0025] Figure 5 For the results of detecting the TelMV disease sample crude extract serially diluted 10-fold by RT-PCR;
[0026] Figure 6 For the detection results of the TelMV disease sample crude extract serially diluted 10-fold by the method combining RT-RAA and CRISPR / Cas12a;
[0027] Figure 7 For the virus-carrying situation of field disease samples detected by the RT-PCR method; in the figure, M is Marker 5000, + is the positive control, - is the negative control, and 1-7 are the numbers of field-collected samples;
[0028] Figure 8To detect the virus-carrying situation of field disease samples based on the RT-RAA and CRISPR / Cas12a visualization system; in the figure, M is Marker 5000, + is the positive control, - is the negative control, and 1-7 are the numbers of field-collected samples. Detailed implementation manners
[0029] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.
[0030] Unless otherwise specified, the test methods used in the following examples are all conventional molecular biology methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.
[0031] Example 1 Screening of RT-RAA reaction primers and reaction conditions
[0032] 1. Screening of RT-RAA reaction primers
[0033] Three pairs of RT-RAA reaction primers were designed according to the Telosma mosaic virus.
[0034] The RT-RAA reaction system was: 1 μL of crude extract of TelMV disease sample, 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of upstream primer (primer concentration: 10 μmol / L), 1 μL of downstream primer (primer concentration: 10 μmol / L), and made up to 25 μL with ddH 2 O; Reacted in a metal bath at 42 °C for 30 min according to the recommended temperature in the kit instructions. After the reaction, the reaction product was treated with a PCR clean-up kit (purchased from Suzhou Youyiland Biotechnology Co., Ltd.) to obtain 20 μL of clean product, and 10 μL was taken for 2% agarose gel electrophoresis analysis to observe the electrophoresis results. As Figure 1 shown, all three pairs of primers could amplify relatively bright electrophoresis bands. Among them, the first pair of primers had the best amplification effect, and the first pair of primers was selected for subsequent experiments. The first pair of primers were named RAA-TelMV-1F and RAA-TelMV-1R respectively, and their nucleotide sequences were as shown in SEQ ID NO.1 and SEQ ID NO.2 in sequence.
[0035] 2. Screening of RT-RAA reaction primer concentration
[0036] To determine the optimal primer concentration required for the reaction, different final primer concentration gradients were set up in the experiment, namely 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, and 0.8 μM. RT-RAA reaction system: 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (10 μmol / L), 1 μL of RAA-TelMV-1R (10 μmol / L), 1 μL of crude TelMV-infected sample extract, and made up to 25 μL with ddH 2 O. Finally, the reaction was carried out in a metal bath at 42 °C for 30 min according to the recommended temperature in the kit instructions. After the reaction, the reaction products were treated with a PCR clean-up kit (purchased from Suzhou Youyiland Biotechnology Co., Ltd.) to obtain 20 μL of clean products. 10 μL was taken and analyzed by 2% agarose gel electrophoresis to observe the electrophoresis results. The results are as Figure 2 shown. Clear target bands could be amplified at primer concentrations of 0.2 μM, 0.4 μM, 0.6 μM, and 0.8 μM. To save primer usage and avoid excessive primer dimers, the concentrations of the forward and reverse primers in the system were determined to be 0.4 μM.
[0037] 3. Screening of RT-RAA reaction temperature
[0038] According to the determined primer concentration, the reaction temperature was explored. Different temperature gradients were set up in the experiment, namely 32 °C, 34 °C, 37 °C, 39 °C, 42 °C, and 44 °C. RT-RAA reaction system: 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (10 μmol / L), 1 μL of RAA-TelMV-1R (10 μmol / L), 1 μL of crude TelMV-infected sample extract, and made up to 25 μL with ddH 2 O. Water bath at different temperatures for 20 min. After the reaction, the reaction products were purified with a PCR clean-up kit, and 5 μL of the purified products was detected by 1% agarose gel electrophoresis.
[0039] The experimental results are as Figure 3 shown. Target bands could be amplified at temperature gradients of 32 °C, 34 °C, 37 °C, and 39 °C. Among them, the best amplification effect could be achieved at 37 °C. To be applicable to the environmental conditions of field detection, the reaction temperature in the system was determined to be 37 °C.
[0040] 4. Screening of RT-RAA reaction time
[0041] The experiment set different reaction time gradients, namely 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min and 45 min. RT-RAA reaction system: 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (10 μmol / L), 1 μL of RAA-TelMV-1R (10 μmol / L), 1 μL of TelMV-infected sample crude extract, and made up to 25 μL with ddH 2 O. Incubated in a water bath at 37 °C for different times. After the reaction, the reaction products were purified using a PCR clean-up kit, and 5 μL of the purified product was detected by 1% agarose gel electrophoresis.
[0042] The experimental results are as Figure 4 shown. Target bands could be amplified at reaction times of 20 min, 25 min, 30 min, 35 min and 45 min. Among them, good amplification effects were achieved at 20, 30 min, 35 min and 45 min, and there was no significant difference in the band brightness after 25 min. To achieve rapid detection, the reaction time in the system was determined to be 20 min.
[0043] In summary, the optimal primer concentration of RT-RAA screened in this experiment was 0.4 μM, the optimal reaction temperature was 37 °C, and the optimal reaction time was 20 min.
[0044] Example 2 Sensitivity Detection
[0045] To verify the sensitivity of the detection method, the concentration of the TelMV-infected sample crude extract was measured using an ultra-micro ultraviolet-visible spectrophotometer, and the initial concentration of the crude extract was determined to be 4.83×10 1 ng / μL. Using this as a template, 10-fold serial dilutions were performed for a total of 7 gradients. The diluted template concentrations were 4.83×10 1 ng / μL, 4.83×10 0 ng / μL, 4.83×10 -1 ng / μL, 4.83×10 -2 ng / μL, 4.83×10 -3 ng / μL, 4.83×10 -4 ng / μL, 4.83×10 -5 ng / μL and 4.83×10 -6 ng / μL. Then, the RT-PCR method and the visualization method were used to detect the crude extracts with different dilution multiples.
[0046] RT-PCR detection system: The RT-PCR reaction was carried out using the HiScript II One Step RT-PCR Kit (DyePlus) from Vazyme. Using the crude extracts of TelMV at different dilution multiples as templates, 1 μL of the crude extract was taken respectively, the reaction volume was 20 μL, and the reaction conditions were: 50 °C for 30 min, 95 °C for 3 min, (95 °C for 30 sec, 56 °C for 30 sec, 72 °C for 30 sec, 30 cycles), 72 °C for 5 min, 12 °C for 5 min, and the reaction was ended. The PCR reaction was carried out on a gradient PCR instrument. The PCR products were detected by 1% agarose gel electrophoresis, and CK was the water control.
[0047] Visual detection system: Using the crude extracts at different dilution multiples as templates, 1 μL of the crude extract was taken respectively, 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (10 μmol / L), 1 μL of RAA-TelMV-1R1 (10 μmol / L), and made up to 25 μL with ddH2O, and incubated in a water bath at 37 °C for 20 min. After the water bath, 2 μL of the RT-RAA reaction product, 0.4 μL of LbCas12a (10 μmol / L), 0.4 μL of TelMV-crRNA (10 μmol / L), 2 μL of ssDNA (10 μmol / L), 2 μL of 10x Enhanced Buffer, and made up to 20 μL with ddH 2 O. Incubated in a water bath at 37 °C for 10 min. After the reaction, the reaction products of different templates were observed for fluorescence under blue light, and CK was the water control. Among them, the nucleotide sequence of EAPV-CrRNA is shown in SEQ ID NO.3; the sequence of the ssDNA reporter molecule is 5'-FAM-CCCCCCCC-BHQ1-3', where FAM is 5-carboxyfluorescein and BHQ1 is a fluorescence quencher; the formula of 10x Enhanced Buffer is: 100 mM Tris-HCl (pH 9.0), 100 mM NaCl, 150 mM MgCl 2 , 10 mM DTT, 5% PEG-200.
[0048] The RT-PCR detection results are as Figure 5 shown. With the increase of the dilution multiple, the brightness of the target band showed a decreasing trend. The detection by RT-PCR was that when the crude extract was diluted to 10 4 times (4.83×10 -2 ng / μL), a weak electrophoresis band could still be detected. The visual detection results are as Figure 6 shown. When the crude extract was diluted 10 7 times (4.83×10-6 After (ng / μL), clear fluorescence signals could still be observed under blue light after RT-RAA amplification. Based on the above results, compared with traditional RT-PCR, the detection system combining RT-RAA and CRISPR / Cas12a significantly enhanced the detection sensitivity.
[0049] Example 3 Field Detection
[0050] Seven samples suspected of being infected with TelMV were collected from the main producing areas of passion fruit in Nanning, Beihai, Baise, Yulin, Guigang, Wuzhou, and Qinzhou in Guangxi and were detected using the traditional RT-PCR detection system and the established visual detection system based on RT-RAA and CRISPR / Cas12a. The diseased sample infected with TelMV stored in the laboratory was used as a positive control, and the healthy passion fruit sample was used as a negative control. RT-PCR detection system: Take the sample to be tested, and extract the total RNA of the leaves using the RNAiso Plus total RNA extraction kit (TaKaRa Bio Inc., Dalian). The total RNA precipitate was dissolved in 50 μL of DEPC-treated water and stored at -20 °C in the refrigerator for later use. The HiScript II One Step RT-PCR Kit (Dye Plus) from Vazyme was used for RT-PCR reaction. The reaction volume was 20 μL, and the reaction conditions were: 50 °C for 30 min, 95 °C for 3 min, (95 °C for 30 sec, 56 °C for 30 sec, 72 °C for 30 sec, 30 cycles), 72 °C for 5 min, 12 °C for 5 min, and the reaction was terminated. The PCR reaction was carried out on a gradient PCR instrument. The PCR products were detected by 1% agarose gel electrophoresis. The crude extract of the TelMV diseased sample was selected as the positive control, the crude extract not infected with TelMV was the negative sample, and CK was the water control.
[0051] Visual detection system: Take the sample to be tested, add the crude extract buffer and steel beads to grind to obtain the crude extract. Take 1 μL of the crude extract, 3 mg of RT-RAA reaction dry powder, 9.5 μL of Buffer A, 2 μL of Buffer B, 1 μL of RAA-TelMV-1F (10 μmol / L), 1 μL of RAA-TelMV-1R (10 μmol / L), and make up to 25 μL with ddH 2 O. Incubate in a water bath at 37 °C for 20 min. After the water bath, take 2 μL of the RT-RAA reaction product, 0.4 μL of LbCas12a (10 μmol / L), 0.4 μL of TelMV-crRNA (10 μmol / L), 2 μL of ssDNA (10 μmol / L), 2 μL of 10x Enhanced Buffer, and make up to the volume with ddH 2Make up to 20 μL. Incubate in a 37 °C water bath for 20 min. After the reaction, observe the fluorescence of the reaction product under blue light. Fluorescence indicates a positive result, and no fluorescence indicates a negative result. Select the crude extract of TelMV-infected samples as the positive control, the crude extract of samples not infected with TelMV as the negative sample, and CK as the water control.
[0052] The RT-PCR detection results are as Figure 7 shown: Obvious electrophoresis bands can be detected in 7 samples suspected of being infected with TelMV; The results of the visual detection system based on RT-RAA and CRISPR / Cas12a are as Figure 8 shown: Clear fluorescence signals can be observed in all 7 samples suspected of being infected with TelMV. The above results indicate that the detection results obtained by using the two detection systems are the same. Monoclonal sequencing of the RT-PCR products shows that the consistency of the obtained sequences with the reference sequences registered in GenBank is above 99%, indicating that the method for visual detection of TelMV based on RT-RAA and CRISPR / Cas12a established in the present invention can be used for rapid detection of field samples, and the results are accurate and reliable.
[0053] The foregoing description of specific exemplary embodiments of the invention has been presented for purposes of illustration and example. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is obvious that many modifications and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A primer set for detecting tuberose mosaic virus based on RT-RAA and CRISPR / Cas12a, characterized in that: Includes RT-RAA amplification primer pair and CRISPR / Cas12a detection primer; The RT-RAA amplification primer pair includes RAA-TelMV-1F and RAA-TelMV-1R, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; The CRISPR / Cas12a detection primer is TelMV-CrRNA, and its nucleotide sequence is shown in SEQ ID NO.
3.
2. Use of the detection primer set according to claim 1 in detecting tuberose mosaic virus.
3. A reagent or kit for detecting tuberose mosaic virus, characterized in that: Contains the detection primer set as claimed in claim 1.
4. The kit according to claim 3, characterized in that: The kit also includes a single LbCas12a protein required for the CRISPR / Cas12a detection system, and a ssDNA reporter molecule for CRISPR / Cas12a detection, wherein the sequence of the ssDNA reporter molecule is 5'-FAM-CCCCCCCC-BHQ1-3', wherein the FAM is 5-carboxyfluorescein, and BHQ1 is a fluorescence quencher.
5. A method for detecting tuberose mosaic virus based on RT-RAA and CRISPR / Cas12a, characterized in that: The nucleic acid of the sample to be tested is used as a template and the kit according to claim 3 or 4 is used for detection.
6. The detection method according to claim 5, characterized in that: The following steps are involved: S1, taking the sample to be tested, adding crude extract buffer and steel balls for grinding to obtain a crude extract; wherein the formula of the crude extract buffer is: 6% PEG200, 20mM NaOH; S2, use the RT-basic nucleic acid amplification kit of Hangzhou Zhongce Biotechnology Co., Ltd. to perform RT-RAA reaction, take 1 μL of crude extract and add it to the RT-RAA reaction system, the RT-RAA reaction system is: RT-RAA reaction powder 3 mg, Buffer A 9.5 μL, Buffer B 2 μL, RAA-TelMV-1F 1 μL, RAA-TelMV-1R 1 μL, and make up 25 μL with ddH2O; 37℃ water bath for 20 min; S3, take 2μL of RT-RAA reaction product and add it to the CRISPR / Cas12a detection system, incubate in a 37℃ water bath for 10min, and observe the fluorescence of the reaction product under blue light after the reaction is completed. Fluorescence is positive, and no fluorescence is negative. The CRISPR / Cas12a detection system is: LbCas12a 0.4μL, EAPV-crRNA 0.4μL, ssDNA 2μL, 10x Enhanced Buffer 2μL, RT-RAA reaction product 2μL, and fill it up to 20μL with ddH2O. The formula of 10x Enhanced Buffer is: 100mM Tris-HCl, 100mM NaCl, 150mM MgCl 2、 10 mM DTT, 5% PEG-200.