CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system for visually detecting bovine viral diarrhea virus and detection method

Abstract

The invention discloses a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system for visually detecting bovine viral diarrhea viruses and a detection method, and belongs to the technical field of virus detection. According to the invention, RT-CPA pre-amplification is combined with Cas12a signal amplification by integrating reverse transcription cross primer amplification (RT-CPA) and targeted cutting capability of CRISPR / Cas12a, so that a bovine viral diarrhea virus detection platform is successfully constructed, and the sensitivity and specificity of BVDV detection are remarkably improved. Compared with traditional RT-PCR, the RT-CPA technology has the advantages that reaction conditions are remarkably simplified through isothermal amplification, dependence of a thermal cycler is avoided, meanwhile, amplification efficiency is improved through cross primer design, and the lower detection limit reaches 8.0 copies / microliter.

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 an important member of the genus Pestivirus in the family Flaviviridae. Its infection can cause clinical symptoms such as diarrhea, fever, abortion and persistent infection (PI) in cattle, causing significant economic losses to the global cattle industry. PI cattle are the main source of infection of the virus. Their secretions and excrement carry the virus for a long time, which has become a key difficulty in the prevention and control of BVDV. Therefore, the development of rapid, sensitive and field-applicable technical means is of great significance for early diagnosis and accurate elimination of PI cattle. At present, the detection of BVDV mainly relies on serological methods (such as ELISA), virus isolation and culture, and nucleic acid detection technology (such as RT-PCR, qRT-PCR). Although RT-PCR and fluorescence quantitative PCR have high sensitivity, and the detection limit of some methods is as low as 5.0267 copies / μL, they rely on precision instruments and professional operations, which are difficult to implement quickly at the grassroots level or on the spot. 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 visualize the results 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 the entire process takes only 1.5 hours, which is significantly better than traditional RT-qPCR. Recent studies have shown that the CRISPR-Cas12a-based platform has a sensitivity of up to 20 copies in BVDV detection, and can cover all known BVDV-1 and BVDV-2 subtypes, which is significantly better than traditional methods. Cross-priming Amplification (CPA) is a new isothermal amplification technology. It can achieve rapid amplification of template DNA at a constant temperature by designing specific primers. This technology is also used in the detection of BVDV, so that 640fg of viral load can be detected under the condition of 62℃ reaction for 60min, further reducing the detection limit and shortening the time. The research method of CPA reverse transcription for virus detection has only been applied in the detection of porcine transmissible gastroenteritis virus, and has shown a high sensitivity. However, it has not been reported in the detection research of other viruses. At the same time, existing research focuses on the optimization of a single technical link. The synergistic mechanism of RT-CPA and CRISPR-Cas12a and its application in complex samples still need to be further explored. 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 the 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 visually detecting bovine viral diarrhea virus, comprising 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, CPA primers, include stripping primer 4s, stripping primer 5a, cross primer 2a1s, detection primer 2a and 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] The third technical solution of the present invention is a method for detecting bovine viral diarrhea virus for non-disease detection or treatment purposes, 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 to detect the amplified product 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, the specific region of the BVDV-E0 gene was first selected as the detection site for the design of CPA primers and crRNA, ensuring the efficient recognition of the target sequence. The reaction conditions were then optimized again, confirming 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, avoids dependence on thermal cyclers, and improves the amplification efficiency through cross-primer design, so that 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0021] Figure 1 The figure shows the screening results of RT-CPA primer pairs, where M: DNA marker; NC: negative control; 1-5: CPA primer sets in sequence.

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

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

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

[0025] Figure 5 This is the result of optimizing the dNTPs concentration in the RT-CPA reaction. M: DNA marker; 1-5: 0.6mmol / L, 0.7mmol / L, 0.8mmol / L, 0.9mmol / L, 1.0mmol / L.

[0026] Figure 6 The figure is the crRNA screening result. 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 figure is the specific detection result. Among them, NC: negative control; 1: bovine coronavirus; 2: bovine rotavirus; 3: bovine viral diarrhea virus; 4: bovine enterovirus; 5: Escherichia coli; 6: Salmonella.

[0029] Fig. 9 This is the sensitivity test result diagram. Among them, 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, 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 embodiment of the present invention also provides a method for detecting bovine viral diarrhea virus for non-disease detection or treatment purposes, 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 to detect the amplified product 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 of stripping primers 4s and 5a each, 2 μL of cross primer 2a1s, 1.6 μL of detection primers 2a and 3a each, 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: reacting at 40° C., collecting fluorescence every 1 min, and reacting for 30 min.

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

[0053] 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. 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, affirming the feasibility of the detection method. Compared with traditional RT-PCR, RT-CPA technology significantly simplifies the reaction conditions by isothermal amplification, avoids thermal cycler dependence, and improves the 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 cumbersome steps of traditional gel electrophoresis, and providing technical support for the application of grassroots veterinary laboratories or farms. By designing multiple groups 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] The present invention successfully constructed an efficient, sensitive and easy-to-operate BVDV detection platform by combining multiple technologies, providing an important tool for early diagnosis and prevention of viral diarrhea. Its technical framework can also be extended to other virus detection fields, and has a wide range of 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 samples were collected from anal swabs from different cattle farms in the surrounding areas of Pingliang, and LbCas12a protease, CleavageBuffer, 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 whole genome sequence of BVDV-1 (EU709763.1) was retrieved from NCBI, species specificity analysis was performed, and 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 labeled 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 standard According to the CPA primer and crRNA design position, the DNA target fragment was synthesized and connected to the PUC57 vector to synthesize the positive plasmid, which was sent to Jinkairui Bioengineering Co., Ltd. for sequencing verification. The copy number of the recombinant plasmid is 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 63°C and the reaction time was 60 min. The CPA primer combinations in Table 2 were screened to determine the primers used to optimize 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, the primer combination 1 has 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) are selected to optimize the overall CPA reaction system.

[0068] 1.4.2 Optimization of RT-CPA reaction time The reaction temperature was set to 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, 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 RT-CPA reaction temperature optimization The optimal reaction temperature of the CPA reaction was 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 CPA reaction temperature was set to 58°C, 60°C, 63°C, 66°C, and 70°C to observe the effect on the amplification reaction. The results are as follows: Figure 3 As shown, the primer amplification effect is better at 58°C, and the reaction temperature of 58°C is selected as the reaction temperature for the next step system optimization.

[0072] 1.4.4 Optimization of primer concentration in RT-CPA reaction The concentrations of the selected primer groups 4s-c, 2a 1s-c, 3a-c, 2a-c, and 5a-c were set according to Table 3, and 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 sets to screen the primer concentrations. The results are as follows: Figure 4 As shown, each group can amplify effective target bands, among which the primer concentration group 4 has the best amplification effect, so 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 systems were 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, 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 was selected as the concentration used in the final reaction system.

[0079] 1.5 Establishment of RT-CPA-CRISPR / Cas12a method The RT-CPA-CRISPR / Cas12a detection system is 30 μL in total, including: Cleavage Buffer 3 μL, LbCas12a 1 μL, crRNA 1 μL, ssDNA 1.2 μL, amplification product 1 μL, ddH2O2 2.8 μL. The reaction was carried out at 40°C, and the fluorescence was collected every 1 min for 30 min. When green fluorescence was observed in the reaction product solution, the bovine viral diarrhea virus test result was determined to be positive.

[0080] 1.5.1 crRNA screening Based on the three groups 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 step of 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 performed on the concentration combinations in pairs 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, the fluorescence signal is strongest when the Cas12a concentration and crRNA concentration are 75nmol / L and 125nmol / L, respectively.

[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 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 gradient dilutions were performed in turn to verify the sensitivity of the RT-CPA-CRISPR / Cas12a detection system. The results are shown in Fig. 9 As shown, when the detection limit is 8.0×10 0 When the detection rate was 2.3449 W / mL, fluorescence signals were still generated, indicating that the established detection system had strong sensitivity.

[0086] 1.5.4 The specificity experiment of RT-CPA-CRISPR / Cas12a detection method 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 negative control to detect the specificity of this 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. The results are as follows Figure 8 As shown, BVDV showed a strong fluorescence value, while other samples and negative controls had no fluorescence values, indicating that the established method has strong specificity.

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

[0089] The 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 consistency rate between the RT-CPA-CRISPR / Cas12a detection method and the conventional PCR detection method can reach 92.86%, with good consistency, indicating that this method has high credibility 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 clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A CRISPR-Cas12a system for visual detection of bovine viral diarrhea virus, characterized in that Includes Cleavage Buffer 3μL, LbCas12a 1μL, crRNA 1μL, ssDNA 1.2μL, amplification product 1μL and ddH2O22.8μL.

2. The CRISPR-Cas12a system according to claim 1, characterized in that The nucleotide sequence of the crRNA is shown in SEQ ID NO.26, and the concentration is 125nmol / L; the concentration of the LbCas12a is 75nmol / L.

3. The CRISPR-Cas12a system according to claim 1, characterized in that The amplification product is the RT-CPA reaction product of the sample to be tested.

4. CPA primer, characterized in that It includes a stripping primer 4s, a stripping primer 5a, a cross primer 2a1s, a detection primer 2a and a detection primer 3a; The nucleotide sequence of the stripping primer 4s is shown in SEQ ID NO.2; The nucleotide sequence of the stripping primer 5a is shown in SEQ ID NO.1; The nucleotide sequence of the cross primer 2a1s is shown in SEQ ID NO.5; The nucleotide sequence of the detection primer 2a is shown in SEQ ID NO.3; The nucleotide sequence of the detection primer 3a is shown in SEQ ID NO.

4.

5. A method for detecting bovine viral diarrhea virus for non-disease detection or treatment purposes, characterized in that: The following steps are involved: (1) amplifying the sample to be tested using the CPA primers described in claim 4 to obtain an amplified product; (2) Using the CRISPR-Cas12a system described in any one of claims 1 to 3 to detect the amplified product to determine whether it contains bovine viral diarrhea virus.

6. The detection method according to claim 5, characterized in that: The amplification reaction system is: 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 of stripping primers 4s and 5a, 2 μL of cross primer 2a1s, 1.6 μL 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.

7. The detection method according to claim 6, characterized in that: 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.

8. The detection method according to claim 5, characterized in that: The amplification reaction conditions are 58° C. for 60 min.

9. The detection method according to claim 5, characterized in that: The detection conditions for detecting the amplified product using the CRISPR-Cas12a system according to any one of claims 1 to 3 are: reacting at 40° C., collecting fluorescence every 1 min, and reacting for 30 min.

10. The detection method according to claim 5, characterized in that: The criterion for judging whether the bovine viral diarrhea virus is contained 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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