A CRISPR-Cas12a system and detection method for visual detection of bovine viral diarrhea virus

Abstract

The present invention discloses a CRISPR-Cas12a system and detection method for visual detection of bovine viral diarrhea virus, which belongs to the field of virus detection technology. The present invention integrates reverse transcription cross primer amplification (RT-CPA) and the targeted cutting ability of CRISPR / Cas12a, combines RT-CPA pre-amplification with Cas12a signal amplification, and successfully constructs a bovine viral diarrhea virus detection platform, which significantly improves the sensitivity and specificity of BVDV detection. Compared with traditional RT-PCR, RT-CPA technology significantly simplifies the reaction conditions through isothermal amplification, avoids dependence on thermal cyclers, and improves amplification efficiency through cross primer design, so that the detection limit reaches 8.0 copies / μL.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a CRISPR-Cas12a system and a detection method for visually detecting bovine viral diarrhea virus. Background Art

[0002] Bovine viral diarrhea virus (BVDV) is a prominent member of the genus Pestivirus in the family Flaviviridae. Infection with BVDV can cause clinical symptoms in cattle, including diarrhea, fever, abortion, and persistent infection (PI), resulting in significant economic losses for the global cattle industry. PI cattle, as the primary source of infection, carry the virus in their secretions and excreta for extended periods, presenting a key challenge in BVDV prevention and control. Therefore, the development of rapid, sensitive, and field-compatible detection methods is crucial for early diagnosis and the precise culling of PI cattle. Currently, BVDV detection relies primarily on serological methods (e.g., ELISA), virus isolation and culture, and nucleic acid detection techniques (e.g., RT-PCR and qRT-PCR). Although RT-PCR and quantitative PCR offer high sensitivity, with some methods achieving detection limits as low as 5.0267 copies / μL, they rely on sophisticated instrumentation and specialized procedures, making rapid implementation difficult at the grassroots level or in the field. In recent years, although loop-mediated isothermal amplification (LAMP) and reverse transcription-recombinase polymerase amplification (RT-RPA) technologies have simplified the nucleic acid amplification steps, they still face limitations such as long time consumption and high false positive rate.

[0003] With the development of molecular diagnostic technology, the CRISPR-Cas12a system has shown significant advantages in the field of nucleic acid detection due to its unique trans-cleavage activity, and has gradually become a research hotspot in the field of pathogen detection. When the Cas12a-crRNA complex recognizes the target DNA, it can non-specifically cut the single-stranded DNA reporter molecule, and the results can be visualized through fluorescence or test strip signals. For example, CRISPR-Cas12a technology has been successfully applied to the detection of African swine fever virus (ASFV) and avian influenza virus (AIV), with a detection limit of up to 6.7 copies / μL and a full process time of only 1.5 hours, which is significantly better than traditional RT-qPCR. Recent studies have shown that the CRISPR-Cas12a-based platform can detect BVDV with a sensitivity of up to 20 copies and can cover all known BVDV-1 and BVDV-2 subtypes, which is significantly better than traditional methods. Cross-priming amplification (CPA) is a novel isothermal amplification technology that rapidly amplifies template DNA at a constant temperature by designing specific primers. This technology has also been used in the detection of BVDV, enabling the detection of 640 fg of viral load at 62°C for 60 minutes, further reducing the detection limit and shortening the time. The CPA reverse transcription method for virus detection has only been applied to the detection of porcine transmissible gastroenteritis virus and has demonstrated high sensitivity. However, it has not been reported in the detection of other viruses. Existing research has mostly focused on optimizing a single technical link, and the synergistic mechanism of RT-CPA and CRISPR-Cas12a and its application in complex samples still require in-depth exploration. Summary of the Invention

[0004] The purpose of the present invention is to provide a CRISPR-Cas12a system and detection method for visual detection of bovine viral diarrhea virus to solve the problems existing in the above-mentioned prior art. The present invention aims to establish a BVDV rapid detection system based on RT-CPA-CRISPR-Cas12a, realize efficient amplification and specific identification of BVDV nucleic acid, combine with a fluorescent reporter system, develop a visual interpretation scheme that does not require complex instruments, and provide technical support for grassroots prevention and control of BVDV.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a CRISPR-Cas12a system for visual detection of bovine viral diarrhea virus, including 3 μL of Cleavage Buffer, 1 μL of LbCas12a, 1 μL of crRNA, 1.2 μL of ssDNA, 1 μL of amplification product and 2.8 μL of ddH2O2.

[0007] The second technical solution of the present invention is a CPA primer, comprising a stripping primer 4s, a stripping primer 5a, a cross primer 2a1s, a detection primer 2a and a detection primer 3a;

[0008] The nucleotide sequence of the stripping primer 4s is shown in SEQ ID NO.2;

[0009] The nucleotide sequence of the stripping primer 5a is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the cross primer 2a1s is shown in SEQ ID NO.5;

[0011] The nucleotide sequence of the detection primer 2a is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the detection primer 3a is shown in SEQ ID NO.4.

[0013] A third technical solution of the present invention is a method for detecting bovine viral diarrhea virus for purposes other than disease detection or treatment, comprising the following steps:

[0014] (1) amplifying the sample to be tested using the CPA primers to obtain an amplified product;

[0015] (2) Using the CRISPR-Cas12a system, the amplified product is detected to determine whether it contains bovine viral diarrhea virus.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] 1. The present invention successfully constructed a bovine viral diarrhea virus detection platform by integrating reverse transcription cross primer amplification (RT-CPA) and the targeted cleavage ability of CRISPR / Cas12a, combining RT-CPA pre-amplification with Cas12a signal amplification, and significantly improving the sensitivity and specificity of BVDV detection.

[0018] 2. In the process of establishing the detection method of the present invention, we first selected a specific region of the BVDV-E0 gene as the detection site for the design of CPA primers and crRNA, ensuring efficient recognition of the target sequence. The reaction conditions were then optimized to confirm the feasibility of the detection method.

[0019] 3. Compared with traditional RT-PCR, the RT-CPA technology provided by the present invention significantly simplifies the reaction conditions through isothermal amplification, avoiding dependence on thermal cyclers. At the same time, the amplification efficiency is improved through cross-primer design, and the detection limit reaches 8.0 copies / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The results of RT-CPA primer pair screening are shown in Figure 1. M: DNA marker; NC: negative control; 1-5: CPA primer sets, respectively.

[0022] Figure 2 This is the result of RT-CPA reaction time optimization. M: DNA marker; 1-5: 15 min, 30 min, 45 min, 60 min, 70 min.

[0023] Figure 3 This is the result of RT-CPA reaction temperature optimization. M: DNA marker; 1-5: 58°C, 60°C, 63°C, 66°C, 70°C.

[0024] Figure 4 This is the result of primer concentration optimization for RT-CPA reaction. M: DNA marker; 1-5: CPA primer concentration combinations.

[0025] Figure 5 This figure shows the optimization results of dNTPs concentrations in the RT-CPA reaction. M: DNA marker; 1-5: 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L.

[0026] Figure 6 The crRNA screening results are shown in Figure 1. A: Fluorescence intensity graph; B: Fluorescence intensity analysis graph; NC: Negative control

[0027] Figure 7 Optimization of crRNA and Cas12a concentrations.

[0028] Figure 8 The results of specificity testing are shown in Figure 1. NC: negative control; 1: bovine coronavirus; 2: bovine rotavirus; 3: bovine viral diarrhea virus; 4: bovine enterovirus; 5: Escherichia coli; 6: Salmonella.

[0029] Figure 9 The sensitivity test results are shown in Figure 1. NC: negative control; 1-8: 8.0×10 7 -8.0×10 0 copies / μL. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] An embodiment of the present invention provides a CRISPR-Cas12a system for visually detecting bovine viral diarrhea virus, including 3 μL of Cleavage Buffer, 1 μL of LbCas12a, 1 μL of crRNA, 1.2 μL of ssDNA, 1 μL of amplification product, and 2.8 μL of ddH2O2.

[0037] In some specific embodiments, the nucleotide sequence of the crRNA is as shown in SEQ ID NO.26, and the concentration is 125nmol / L; the concentration of the LbCas12a is 75nmol / L.

[0038] In some specific embodiments, the amplification product is a RT-CPA reaction product of the sample to be tested.

[0039] The embodiment of the present invention also provides CPA primers, including stripping primer 4s, stripping primer 5a, cross primer 2a1s, detection primer 2a and detection primer 3a;

[0040] The nucleotide sequence of the stripping primer 4s is shown in SEQ ID NO.2;

[0041] The nucleotide sequence of the stripping primer 5a is shown in SEQ ID NO.1;

[0042] The nucleotide sequence of the cross primer 2a1s is shown in SEQ ID NO.5;

[0043] The nucleotide sequence of the detection primer 2a is shown in SEQ ID NO.3;

[0044] The nucleotide sequence of the detection primer 3a is shown in SEQ ID NO.4.

[0045] The present invention also provides a method for detecting bovine viral diarrhea virus for purposes other than disease detection or treatment, comprising the following steps:

[0046] (1) amplifying the sample to be tested using the CPA primers to obtain an amplified product;

[0047] (2) Using the CRISPR-Cas12a system, the amplified product is detected to determine whether it contains bovine viral diarrhea virus.

[0048] In some specific embodiments, the amplification reaction system is: 2 μL of buffer, 0.4 μL of MgSO4, 1 μL of BstDNA polymerase, 4 μL of betaine, 1.6 μL of dNTPs, 0.4 μL each of stripping primers 4s and 5a, 2 μL of cross primer 2a1s, 1.6 μL each of detection primers 2a and 3a, 0.5 μL of M-MLV reverse transcriptase, 1 μL of template RNA, and ddH2O added to 20 μL.

[0049] In some specific embodiments, the concentration of the stripping primer 4s and the stripping primer 5a is 0.2 μmol / L; the concentration of the cross primer 2a1s is 1 μmol / L; the concentration of the detection primer 2a and the detection primer 3a is 0.8 μmol / L; and the concentration of the dNTPs is 0.8 mmol / L.

[0050] In some specific embodiments, the amplification reaction condition is 58° C. for 60 min.

[0051] In some specific embodiments, the detection conditions for detecting the amplified product using the CRISPR-Cas12a system are: reaction at 40° C., collecting fluorescence every 1 minute, and reacting for 30 minutes.

[0052] In some specific embodiments, the criterion for determining whether the solution contains bovine viral diarrhea virus is: when green fluorescence is observed in the reaction product solution, the bovine viral diarrhea virus test result can be determined to be positive.

[0053] The present invention integrates the targeted cutting ability of reverse transcription cross primer amplification (RT-CPA) and CRISPR / Cas12a, combines RT-CPA pre-amplification with Cas12a signal amplification, and successfully constructs a bovine viral diarrhea virus detection platform, which significantly improves the sensitivity and specificity of BVDV detection. In the process of method establishment, the specific region of the BVDV-E0 gene is first selected as the detection site to design CPA primers and crRNA, ensuring efficient recognition of the target sequence, and optimizing the reaction conditions again, which confirms the feasibility of the detection method. Compared with traditional RT-PCR, RT-CPA technology significantly simplifies the reaction conditions by isothermal amplification, avoids dependence on thermal cyclers, and improves amplification efficiency by cross primer design, so that the detection limit reaches 8.0 copies / μL. In addition, the detection specificity is further enhanced by the cascade reaction of CRISPR / Cas12a. By designing specific crRNA to target the DNA of BVDV, cross-reactions with common diarrheal pathogens such as Bcov, BRV, and E. coli are avoided. Compared with electrochemical immunosensors (specificity depends on antibodies), nucleic acid-targeted CRISPR technology is less susceptible to antigenic variation. Its side chain cleavage activity is visualized through fluorescent signals, achieving the ultimate goal from amplification to detection, avoiding the tedious steps of traditional gel electrophoresis, and providing technical support for application in grassroots veterinary laboratories or farms. By designing multiple sets of crRNA, BVDV typing detection kits can be developed, and they can also be detected simultaneously with other co-infected pathogens (such as bovine coronavirus and bovine infectious rhinotracheitis virus). At the same time, it can be extended to the detection of other RNA viruses such as foot-and-mouth disease virus (FMDV), which can effectively promote the healthy development of animal husbandry.

[0054] By combining multiple technologies, this study successfully developed a highly efficient, sensitive, and easy-to-use BVDV detection platform, providing an important tool for the early diagnosis and prevention of viral diarrhea. The technical framework can also be expanded to other viral detection fields, with broad clinical applications and scientific research value.

[0055] Example 1

[0056] 1 Materials and Methods

[0057] 1.1 Main reagents and instruments The bovine viral diarrhea virus (BVDV), bovine coronavirus (Bcov), bovine rotavirus (BRV), bovine enterovirus (Bev), Escherichia coli, and Salmonella used in this experiment were provided by the Gansu Institute of Animal Husbandry and Veterinary Medicine; the diseased material samples were collected from anal swabs of different cattle farms in the surrounding areas of Pingliang; LbCas12a protease, Cleavage Buffer, Bst DNA polymerase, betaine, dNTPs, MgSO4, one-step RT-PCR kit, plasmid extraction kit, and RNA extraction kit were all purchased from Gansu Xirui Biotechnology Co., Ltd.

[0058] 1.2 Design and synthesis of CPA, crRNA, and ssDNA primers The BVDV-1 full genome sequence (EU709763.1) was retrieved from NCBI, and species specificity analysis was performed. The EO gene-specific region was selected as the detection site for the design of CPA primers and crRNA. A fluorescent group FAM was connected to the 5' end of the basic sequence TTATT, and a quenching group BHQⅠ was connected to the 3' end to construct an ssDNA fluorescent labeling probe. The above primers and probes were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., as shown in Table 1.

[0059] Table 1 CPA amplification primers, crRNA sequences and ssDNA probes

[0060]

[0061]

[0062] 1.3 Construction of plasmid standards Based on the CPA primers and crRNA design positions, the DNA target fragment was synthesized and ligated to the PUC57 vector to synthesize a positive plasmid, which was then sent to Jinkairui Bioengineering Co., Ltd. for sequencing verification. The recombinant plasmid copy number was 10 9 The recombinant plasmid was used as a template for subsequent experiments and stored at –20°C.

[0063] 1.4 RT-CPA reaction system A total of 20 μL of RT-CPA reaction system was established, including: 2 μL of buffer, 0.4 μL of MgSO4, 1 μL of Bst DNA polymerase, 4 μL of betaine, 1.6 μL of dNTPs, 0.4 μL each of stripping primers 4s (0.2 μmol / L) and 5a (0.2 μmol / L), 2 μL of cross primer 2a1s (1 μmol / L), 1.6 μL each of detection primers 2a (0.8 μmol / L) and 3a (0.8 μmol / L), 0.5 μL of M-MLV reverse transcriptase, 1 μL of template RNA, and ddH2O added to 20 μL.

[0064] 1.4.1 RT-CPA Primer Screening Based on the above reaction system, the reaction temperature was selected as 63°C and the reaction time was 60 min. The CPA primer combinations in Table 2 were screened to determine the primers optimized for the CPA reaction system.

[0065] Table 2 CPA amplification primer combinations

[0066]

[0067] According to the RT-CPA reaction system, 5 groups of CPA amplification primer combinations were screened, and the results were as follows Figure 1 As shown, primer combination 1 had a better amplification effect, that is, 4s-c (SEQ ID NO.2), 2a 1s-c (SEQ ID NO.5), 3a-c (SEQ ID NO.4), 2a-c (SEQ ID NO.3), and 5a-c (SEQ ID NO.1) were selected to optimize the overall CPA reaction system.

[0068] 1.4.2 Optimization of RT-CPA reaction time The reaction temperature was set at 63°C, and the primer sets of 4s-c, 2a 1s-c, 3a-c, 2a-c, and 5a-c were used to test the optimal reaction time of the CPA reaction at 15 min, 30 min, 45 min, 60 min, and 70 min.

[0069] In the experiment with different reaction times, the reaction time was set to 15min, 30min, 45min, 60min, and 70min. The results are as follows: Figure 2 As shown in the figure, the target band can be amplified at 60 min and 75 min, and the amplification effect is best at 60 min. Therefore, the reaction time of 60 min is selected as the reaction time for the next step system optimization.

[0070] 1.4.3 Optimization of RT-CPA reaction temperature The optimal reaction temperatures of the CPA reaction were determined at 58°C, 60°C, 63°C, 66°C, and 70°C within the optimal reaction time.

[0071] In the experiment with different reaction temperatures, the effects of setting the CPA reaction temperature to 58°C, 60°C, 63°C, 66°C, and 70°C on the amplification reaction were observed. The results are as follows: Figure 3 As shown in the figure, the primer amplification effect is better at 58℃, so the reaction temperature of 58℃ is selected as the reaction temperature for the next step system optimization.

[0072] 1.4.4 Optimization of primer concentrations for RT-CPA reaction The selected primer sets 4s-c, 2a 1s-c, 3a-c, 2a-c, and 5a-c were set at different primer set concentrations according to Table 3. The reaction was carried out at 58°C for 60 min to determine the optimal primer concentration combination for the CPA reaction.

[0073] Table 3 CPA primer concentration

[0074]

[0075]

[0076] Five primer concentration combinations were set for the selected primer groups to screen the primer concentration. The results are as follows: Figure 4 As shown, each group can amplify the effective target band, among which the primer concentration group 4 has the best amplification effect. Therefore, primer concentration group 4 is selected as the next system to optimize the reaction temperature.

[0077] 1.4.5 Optimization of dNTPs concentration in RT-CPA reaction The above reaction system was integrated and the dNTPs concentrations were set to 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L and 1.0 mmol / L for screening.

[0078] Combined with the above optimized reaction system, different concentrations of dNTPs were screened. The results are as follows Figure 5 As shown in the figure, dNTPs at different concentrations can amplify effective target bands, and the amplification effect is best when the dNTPs concentration is 0.8mmol / L and 0.9mmol / L. After comprehensive consideration, the concentration of 0.8mmol / L is selected as the final reaction system concentration.

[0079] 1.5 Establishment of the RT-CPA-CRISPR / Cas12a Method: The RT-CPA-CRISPR / Cas12a detection system (30 μL) consists of: 3 μL Cleavage Buffer, 1 μL LbCas12a, 1 μL crRNA, 1.2 μL ssDNA, 1 μL amplification product, and 2.8 μL ddH2O2. The reaction was incubated at 40°C for 30 minutes, with fluorescence collected every 1 minute. Green fluorescence was observed in the reaction product solution, indicating a positive result for BVDV.

[0080] 1.5.1 crRNA Screening Based on the three sets of amplicon-generating primers screened by CPA primers, six crRNAs were designed and synthesized at the primer positions, and then screened according to the RT-CPA-CRISPR / Cas12a detection system.

[0081] 6 pairs of crRNA fluorescence values ​​are as follows Figure 6 As shown, compared with the control group, the fifth group (cr5 as shown in SEQ ID NO.26) had the strongest fluorescence value and the highest cutting efficiency, followed by cr4 and cr6. crRNA5 and the corresponding primer group were selected for the next experimental verification.

[0082] 1.5.2 Optimization of crRNA and Cas12a concentrations The concentrations of Cas12a and crRNA were set to 25 nmol / L, 50 nmol / L, 75 nmol / L, 100 nmol / L, 125 nmol / L and 150 nmol / L, respectively. Orthogonal experiments were then performed on the concentration combinations to screen out the optimal reaction concentrations of crRNA and Cas12a.

[0083] In order to optimize the optimal reaction concentration of the cleavage system, the concentrations of Cas12a and crRNA were set to 25nmol / L, 50nmol / L, 75nmol / L, 100nmol / L, 125nmol / L and 150nmol / L for orthogonal experiments. The results are as follows Figure 7 As shown, when the Cas12a concentration and crRNA concentration were 75 nmol / L and 125 nmol / L, respectively, the fluorescence signal was the strongest.

[0084] 1.5.3 RT-CPA-CRISPR / Cas12a detection method sensitivity experiment The target concentration was increased from the original concentration to 8×10 7 -8×10 0 The samples were diluted 10-fold with 400 copies / μL, and the sensitivity was verified by CPA-CRISPR / Cas12a detection method.

[0085] The target concentration was set at 8.0×10 7 -8.0×10 0 The 10-fold serial dilutions were performed to verify the sensitivity of the RT-CPA-CRISPR / Cas12a detection system. Figure 9 As shown, when the detection limit is 8.0×10 0 When the detection rate was 2.5447 W / μL, fluorescence signals were still generated, indicating that the established detection system had strong sensitivity.

[0086] 1.5.4 RT-CPA-CRISPR / Cas12a detection method specificity experiment selected bovine viral diarrhea virus (BVDV), bovine coronavirus (BCOV), bovine rotavirus (BRV), bovine norovirus (BNOV), bovine enterovirus (BEV) positive plasmids and Escherichia coli (E. coli) and Salmonella (Salmonella) DNA as detection templates, and used nuclease-free water as a negative control to test the specificity of the method.

[0087] The above optimized results were used to amplify the positive plasmids and DNA of bovine coronavirus, bovine rotavirus, bovine viral diarrhea virus, bovine enterovirus, Escherichia coli, and Salmonella. Figure 8 As shown in the figure, BVDV showed a strong fluorescence value, while other samples and negative controls had no fluorescence value, indicating that the established method has strong specificity.

[0088] 1.6 Clinical Sample Testing: 42 clinical diarrhea samples collected from areas surrounding Pingliang City were processed on-site and stored at -80°C. The established RT-CPA-CRISPR / Cas12a detection method and conventional PCR method were used for control testing. The test results were recorded and the detection method was evaluated.

[0089] 42 diarrheal samples collected clinically were tested for BVDV using the PCR method. At the same time, the 16 positive samples detected were retested using the established RT-CPA-CRISPR / Cas12a detection method, and all of them were positive. The results are shown in Table 4. The concordance rate between the RT-CPA-CRISPR / Cas12a detection method and conventional PCR detection reached 92.86%, with good consistency, indicating that this method is highly reliable and can be used for high-quality detection of clinical samples.

[0090] Table 4 Comparison of clinical sample test results

[0091]

[0092] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting bovine viral diarrhea virus for non-disease detection or treatment purposes, characterized in that: The following steps are involved: (1) Using CPA primers to amplify the sample to be tested to obtain an amplified product; (2) Use the CRISPR-Cas12a system to detect the amplified product to determine whether it contains bovine viral diarrhea virus; The CPA primers include stripping primer 4s, stripping primer 5a, cross primer 2a1s, detection primer 2a and detection primer 3a; The nucleotide sequence of the stripping primer 4s is shown in SEQ ID NO.13; The nucleotide sequence of the stripping primer 5a is shown in SEQ ID NO.12; The nucleotide sequence of the cross primer 2a1s is shown in SEQ ID NO.16; The nucleotide sequence of the detection primer 2a is shown in SEQ ID NO.14; The nucleotide sequence of the detection primer 3a is shown in SEQ ID NO.15; The amplification product is the RT-CPA reaction product of the sample to be tested; The CRISPR-Cas12a system includes 3 μL of Cleavage Buffer, 1 μL of LbCas12a, 1 μL of crRNA, 1.2 μL of ssDNA, 1 μL of amplified product, and 22.8 μL of ddH2O; The nucleotide sequence of the crRNA is shown in SEQ ID NO.26, and the concentration is 125 nmol / L; the concentration of the LbCas12a is 75 nmol / L; the nucleotide sequence of the ssDNA is shown in SEQ ID NO.28, and a fluorescent group FAM is connected to the 5' end and a quenching group BHQ Ⅰ is connected to the 3' end.

2. The detection method according to claim 1, wherein The amplification reaction system is as follows: 2 μL of buffer, 0.4 μL of MgSO4, 1 μL of Bst DNA polymerase, 4 μL of betaine, 1.6 μL of dNTPs, 0.4 μL each of stripping primers 4s and 5a, 2 μL of cross primer 2a1s, 1.6 μL each of detection primers 2a and 3a, 0.5 μL of M-MLV reverse transcriptase, 1 μL of template RNA, and ddH2O added to 20 μL.

3. The detection method according to claim 2, characterized in that The concentrations of the stripping primer 4s and the stripping primer 5a were 0.2 μmol / L; the concentration of the cross primer 2a1s was 1 μmol / L; the concentrations of the detection primer 2a and the detection primer 3a were 0.8 μmol / L; and the concentration of the dNTPs was 0.8 mmol / L.

4. The detection method according to claim 1, wherein The amplification reaction conditions are 58°C for 60 min.

5. The detection method according to claim 1, wherein The detection conditions for detecting the amplified product using the CRISPR-Cas12a system according to claim 1 are: reaction at 40°C, collecting fluorescence every 1 minute, and reacting for 30 minutes.

6. The detection method according to claim 1, characterized in that The criterion for judging whether the solution contains bovine viral diarrhea virus is: when green fluorescence is observed in the reaction product solution, the bovine viral diarrhea virus test result can be determined to be positive.

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