A kit for visual detection of epidemic hemorrhagic disease virus, its usage method and application

Abstract

A kit for visually detecting epidemic hemorrhagic disease virus, and its use method and application, belong to the field of biological detection technology. In order to solve the lack of efficient, highly sensitive and highly specific methods for rapid on-site detection of EHDV in the prior art, the present invention provides a kit for visually detecting epidemic hemorrhagic disease virus, the kit comprising RT-EAR primers and crRNA, as well as RT-ERA amplification system and CRISPR-Cas12a detection system; the RT-ERA primers can specifically amplify target sequences, and the CRISPR-Cas12a detection system has good sensitivity and high specificity. The kit for visually detecting epidemic hemorrhagic disease virus provided by the present invention provides a new detection method for EHDV nucleic acid detection, and the kit can be used for laboratory detection of EHDV and clinical sample analysis, on-site detection, daily monitoring and the like in farms.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a kit for detecting epidemic hemorrhagic disease virus and a using method and application thereof. Background Art

[0002] Epizootic hemorrhagic disease (EHD) is a worldwide epidemic caused by the Epizootic hemorrhagic disease virus (EHDV), a vector-transmitted insect of the genus Culicoides that infects a wide range of wild and domestic ruminants. Ruminants, including deer, cattle, sheep, and alpacas, are susceptible to EHDV, with deer and cattle being the most vulnerable. In white-tailed deer, EHDV infection can cause morbidity as high as 80%, with mortality ranging from 20% to 80%. Infected cattle and sheep can develop severe hemorrhagic disease, characterized by fever, loss of appetite, facial edema, and conjunctival and oral mucosal congestion. Certain serotypes can cause miscarriage and stillbirth in pregnant animals, resulting in significant economic losses.

[0003] The EHDV virion is approximately 80 nm in diameter, exhibits icosahedral symmetry, and lacks an envelope. Its genome consists of 10 segmented double-stranded RNA (dsRNA) strands encoding seven structural proteins (VP1-VP7) and four nonstructural proteins (NS1, NS2, and NS3 / NS3A). VP1, VP4, and VP6 form the inner replicase complex, VP3 and VP7 form the inner virion capsid, and VP2 and VP5 form the outer virion capsid. VP7 induces group-specific antibodies, while VP2 induces virus-specific neutralizing antibodies in the host, which determine EHDV serotype. Currently, at least 10 EHDV serotypes have been identified: EHDV-1, 2, 4, 5, 6, 7, 8, 9, 10, and 11. There is no cross-protection between serotypes.

[0004] Currently, EHDV detection methods include virus isolation and identification, serological testing, and nucleic acid testing. Among them, virus isolation and identification is the "gold standard", but it has high requirements for laboratory personnel, requires professional training, and is time-consuming. Except for laboratory testing, it is rarely used in the field. Serological testing is currently recognized as a universal inspection method in epidemiological surveys, customs import and export quarantine, animal husbandry, etc., but the operation is complicated and time-consuming, and is also not suitable for field application. RT-PCR and fluorescent RT-PCR detection methods have become the preferred methods for laboratory testing due to their high sensitivity, simple operation, and short time consumption, but they require expensive professional equipment and are also not suitable for field testing.

[0005] Therefore, those skilled in the art are eager to develop an efficient, highly sensitive, and highly specific on-site rapid detection method for EHDV that can obtain visual results without relying on precise laboratory instruments. Summary of the Invention

[0006] The present invention solves the problem in the prior art of lacking an efficient, highly sensitive and highly specific method for rapid on-site detection of EHDV and provides a kit for visually detecting EHDV and a method and application thereof.

[0007] One of the objects of the present invention is to provide a kit for visual detection of epidemic hemorrhagic disease virus, the kit comprising an RT-EAR primer and crRNA; the forward primer nucleotide sequence of the RT-ERA primer is shown in SEQ ID NO.8, and the reverse primer nucleotide sequence is shown in SEQ ID NO.11; the crRNA comprises: crRNA1 with a nucleotide sequence shown in SEQ ID NO.1 and crRNA2 with a nucleotide sequence shown in SEQ ID NO.2.

[0008] In a preferred embodiment of the present invention, the RT-ERA primers and crRNA are based on the conserved sequence fragment of the EHDV genome segment S1 as a template.

[0009] In a preferred embodiment of the present invention, the amplification system of the RT-ERA primer in the kit is: 4 μL of solvent, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of RNA template of the sample to be tested, 3.6 μL of DEPC water, and finally 0.4 μL of activator.

[0010] In a preferred embodiment of the present invention, the kit further includes a CRISPR-Cas12a detection system, which includes: Cas12a 75 ng, 10 μM crRNA 0.4 μL, 10 μM ssDNA FQ reporter gene or ssDNA biotin-labeled nucleic acid probe 1 μL, Recombinant RNase Inhibitor 0.4 μL, 10×Cas12aBuffer 1 μL, RT-ERA amplification product to be detected 1-2 μL, and DEPC water to supplement the system to 10 μL.

[0011] A second object of the present invention is to provide a method for using the above-mentioned kit for non-diagnostic purposes, comprising the following steps: adding the sample RNA to be tested to the RT-ERA system for isothermal amplification, reacting at 40°C for 20 minutes, and obtaining an RT-ERA amplification product; adding the above-mentioned RT-ERA amplification product to a CRISPR-Cas12a detection system containing crRNA for reaction, reacting at 37°C for 20 minutes, obtaining a reactant, and then performing result detection on the above-mentioned reactant.

[0012] In a preferred embodiment of the present invention, the method of detecting the result in the method of use includes: real-time fluorescence quantitative PCR analysis, blue light gel cutting instrument analysis or lateral flow test strip analysis.

[0013] In a preferred embodiment of the present invention, the detection step of the real-time fluorescence quantitative PCR analysis is: performing a CRISPR-Cas12a detection reaction in a real-time fluorescence quantitative PCR instrument, and setting the fluorescence value to be read once every 30 seconds, and performing result detection according to the read fluorescence value.

[0014] In a preferred embodiment of the present invention, the detection step of the blue light gel cutting instrument analysis is: placing the reactant obtained after the CRISPR-Cas12a detection reaction is completed under a blue light gel cutting instrument for observation, taking pictures and recording the brightness and color changes of the reaction tube, when the reaction tube appears a fluorescent signal under the blue light gel cutting instrument, it indicates that the test result is positive; when the reaction tube does not appear a fluorescent signal under the blue light gel cutting instrument, it indicates that the test result is negative.

[0015] In a preferred embodiment of the present invention, the detection step of the lateral flow test strip analysis is: placing the reactant obtained after the CRISPR-Cas12a detection reaction on a lateral flow test strip for interpretation of the test results. When the test line T line and the control line C line appear on the test strip at the same time, it indicates that the test result is positive; when only the control line C line appears on the test strip, it indicates that the test result is negative.

[0016] The third object of the present invention is to provide the use of the above-mentioned kit in visual detection of epidemic hemorrhagic disease virus.

[0017] Beneficial effects of the present invention: The present invention provides a kit for visually detecting epidemic hemorrhagic disease virus, and its use method and application. The kit (RT-ERA / CRISPR-Cas12a system) includes RT-EAR primers and crRNA, as well as an RT-ERA amplification system and a CRISPR-Cas12a detection system. The kit for visually detecting epidemic hemorrhagic disease virus provided by the present invention has good sensitivity, specificity and universality; first, the detection limit of the fluorescence signal reading and the fluorescence signal visualization is 1.7×10 1 copies / μL, and the lower limit of detection of the lateral flow test strip visualization was 1.7×10 2 copies / μL; secondly, the above-mentioned RT-ERA / CRISPR-Cas12a system can complete the specific detection of EHDV nucleic acid, and has no cross-reaction with other ruminant arboviral infectious pathogens BTV, AKAV, CHUV and common ruminant viral infectious pathogens PPRV, GPV, BRSV, BRV, BPIV3 and IBRV; finally, the above-mentioned RT-ERA / CRISPR-Cas12a system can complete the nucleic acid detection of at least 8 EHDV serotypes.

[0018] The results of fluorescent RT-PCR laboratory tests on clinically collected blood or tissue samples showed that the test results of the visual detection kit for epidemic hemorrhagic disease virus (RT ERA / CRISPR-Cas12a system) provided by the present invention were consistent with the results of fluorescent RT-PCR laboratory tests, indicating that the detection method provided by the present invention can achieve efficient and visual laboratory tests and is suitable for laboratory testing. The test results based on nucleic acid release and RT ERA / CRISPR-Cas12a system showed that the test results of the visual detection kit for epidemic hemorrhagic disease virus (RT ERA / CRISPR-Cas12a system) provided by the present invention were consistent with the results of fluorescent RT-PCR laboratory tests, indicating that the above-mentioned detection method can obtain accurate visual detection results without relying on precision instruments and is suitable for on-site detection work.

[0019] The kit (RT ERA / CRISPR-Cas12a system) for visual detection of epidemic hemorrhagic disease virus provided by the present invention overcomes the defects of high requirements for the detection environment, long detection cycle, expensive detection instruments, equipment, reagents and consumables, inapplicability to on-site detection, and relatively high false negative rate in the early stage of pathogen infection compared with the pathogen isolation, serological detection and nucleic acid detection technologies in the prior art; the RT ERA / CRISPR-Cas12a system provided by the present invention overcomes the defects of relatively low specificity and sensitivity of the LAMP and RPA isothermal amplification technologies in the prior art, and the LAMP isothermal amplification technology requires at least two pairs of primers to complete the detection, and dimers are easily formed between the primers, reducing the detection efficiency, compared with the LAMP and RPA isothermal amplification technologies in the prior art; the RT ERA / CRISPR-Cas12a system provided by the present invention improves the specificity of the detection reaction compared with the method of separately using RT-ERA isothermal amplification or CRISPR-Cas12a system for detection in the prior art; the RT ERA / CRISPR-Cas12a system provided by the present invention reduces the in vitro transcription step in the detection process compared with the CRISPR-Cas13a system in the prior art, not only reducing the detection cost, but also improving the detection efficiency.

[0020] The RT ERA / CRISPR-Cas12a system provided by the present invention provides a new detection method for EHDV nucleic acid detection, which can be used for clinical sample analysis, on-site detection, daily monitoring, etc. in farms, providing an efficient, convenient and visual technical platform for grass-roots detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a fluorescence reading result diagram for the screening of crRNA in Example 1; the vertical coordinate of Fluorescence is the fluorescence signal intensity, and the horizontal coordinate of Cycles is the number of cycles; NC is the control group;

[0022] Figure 2 It is a screening result diagram of RT-ERA primers in Example 2; A is the screening result diagram of the upstream primer, and B is the screening result diagram of the downstream primer;

[0023] Figure 3 It is a sensitivity detection result diagram of the RT-ERA / CRISPR-Cas12a detection method in Example 2; A is the fluorescence reading result diagram, B is the detection result diagram by a blue light gel cutter, and C is the detection result diagram by a lateral flow detection test strip;

[0024] Figure 4Figure 2 shows the specificity detection results of the RT-ERA / CRISPR-Cas12a detection method in Example 2; A shows the fluorescence reading results, B shows the blue light gel cutting instrument detection results, and C shows the lateral flow test strip detection results;

[0025] Figure 5 This is a diagram showing the RT-ERA / CRISPR-Cas12a validation results for EHDV in Example 2;

[0026] Figure 6 This is the visual detection result of clinical samples in the laboratory of the RT-ERA / CRISPR-Cas12a detection method in Example 4;

[0027] Figure 7 This is a comparison of the results of RT-ERA / CRISPR-Cas12a detection based on TRIzol RNA extraction and laboratory fluorescent RT-PCR detection in Example 4;

[0028] Figure 8 This is a comparison of the results of RT-ERA / CRISPR-Cas12a-test strip detection and laboratory fluorescent RT-PCR detection based on TRIzol RNA extraction in Example 4;

[0029] Figure 9 This is a comparison chart of the results of RT-ERA / CRISPR-Cas12a detection based on nucleic acid-released RNA and laboratory fluorescent RT-PCR detection in Example 4. DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0032] HSDNA Polymerase was purchased from Baori Biotechnology Co., Ltd.;

[0033] Recombinant RNase Inhibitor was purchased from Baori Biotechnology Co., Ltd.;

[0034] GeneJET gel extraction kit was purchased from Thermo Fisher Scientific;

[0035] T7 High Yield RNA Transcription Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd.;

[0036] RT-based nucleic acid amplification reagent (ERA method) was purchased from Suzhou Xianda Gene Technology Co., Ltd.;

[0037] Lateral flow cytometry test strips (CRISPR) were purchased from Suzhou Xianda Gene Technology Co., Ltd.;

[0038] Nucleic acid release agent was purchased from Suzhou Xianda Gene Technology Co., Ltd.;

[0039] GenCRISPR TM Cas12a (Cpf1) Nuclease was purchased from GenScript Biotech Co., Ltd.;

[0040] crRNA was synthesized by GenScript Biotech Co., Ltd.;

[0041] DNA sequence synthesis was performed by Beijing Ruiboxingke Biotechnology Co., Ltd.;

[0042] In the following examples, the present invention provides a visual detection kit for detecting epidemic hemorrhagic disease virus, referred to as the RT-ERA / CRISPR-Cas12a system.

[0043] Example 1: Design and screening of crRNA

[0044] (1) Design of crRNA

[0045] In this example, 8 serotypes of EHDV strains were selected from GenBank as reference sequences, including EHDV-1, EHDV-2, EHDV-4, EHDV-5, EHDV-6, EHDV-7, EHDV-8, and EHDV-10 reference sequences (no EHDV-9 and EHDV-11 reference sequences were available in GenBank); by sequence alignment analysis, the conserved sequence fragment of the EHDV genome segment S1 was selected as the target, and crRNA was designed according to the PAM site, namely crRNA1 (nucleotide sequence shown in SEQ ID NO.1) and crRNA2 (nucleotide sequence shown in SEQ ID NO.2), and the crRNA sequences are shown in Table 1.

[0046] Table 1

[0047]

[0048] Note: Y represents the degenerate base C+U; S represents the degenerate base G+C.

[0049] (2) Preparation of DNA template

[0050] In this example, the EHDV genomic segment S1 sequence with a T7 promoter was synthesized and cloned into the pUC57 vector. The PCR reaction volume was 50 μL, consisting of 5× PrimeSTAR Buffer (10 μL), 2.5 mM dNTP Mixture (4 μL), forward primer (1 μL, final concentration 0.2 μM), reverse primer (1 μL, final concentration 0.2 μM), PrimeSTAR HSDNA Polymerase (0.5 μL), template (1 μL, 50 ng), and ddH2O (32.5 μL). The PCR product was purified using a GeneJET gel recovery kit and used as a DNA template for downstream reactions.

[0051] (3) Screening of crRNA

[0052] In this example, the cleavage efficiency of the crRNA-mediated CRISPR-Cas12a system was evaluated. The crRNA1 (nucleotide sequence shown in SEQ ID NO.1) and crRNA2 (nucleotide sequence shown in SEQ ID NO.2) obtained in step (1) were mixed with crRNA1 and crRNA2 in an equimolar ratio to obtain three test samples. The three test samples were incubated with the EHDV S1 DNA template obtained in step (2) to detect the cleavage efficiency of the CRISPR-Cas12a detection system. The test results were read by a real-time fluorescence quantitative PCR instrument. The reaction temperature was 37°C, and data were recorded every 30 seconds. A fluorescence data graph was prepared to analyze the crRNA efficiency. The CRISPR-Cas12a detection system had a reaction volume of 10 μL and consisted of Cas12a (75 ng), crRNA (0.4 μL, final concentration 400 nM), ssDNA FQ reporter gene (1 μL, final concentration 1 μM), Recombinant RNase Inhibitor (0.4 μL), 10×Cas12a Buffer (1 μL), DNA template (1 μL) and DEPC water (replenish the system to 10 μL).

[0053] The results are as follows Figure 1As shown, the real-time fluorescence quantitative PCR detection results of the crRNA1 and crRNA2 separate experimental groups and the equimolar mixture experimental group all produced fluorescent signals. Compared with the crRNA2 experimental group alone and the equimolar mixture experimental group, the experimental group applying crRNA1 alone produced a higher fluorescence signal value. Therefore, crRNA1 (nucleotide sequence is shown in SEQ ID NO.1) mediated the CRISPR-Cas12a system with higher cutting efficiency, and crRNA1 was selected for subsequent experiments.

[0054] Example 2: Construction and verification of RT ERA / CRISPR-Cas12a detection system

[0055] (1) Design and screening of isothermal amplification primers

[0056] In this example, 6 upstream primers and 8 downstream primers were designed based on the crRNA1 obtained in Example 1 (nucleotide sequence shown in SEQ ID NO. 1) for cross-pairing screening. The primer sequences are shown in Table 2. The RT-ERA product to be tested was obtained by RT-ERA amplification reaction. The RT-ERA amplification reaction volume was 10 μL, including a solvent (4 μL), a forward primer (0.5 μL, a final concentration of 500 nM), a reverse primer (0.5 μL, a final concentration of 500 nM), an EHDV RNA template (1 uL), and DEPC water (3.6 μL). Finally, an activator (0.4 μL) was added. The solvent was provided in a commercial RT-ERA kit. The RT-ERA amplification reaction conditions were 40 ° C for 20 min. The RT-ERA products of the above-mentioned different primer combinations were drawn for use in the CRISPR-Cas12a detection system, which had a reaction volume of 10 μL and consisted of Cas12a (75 ng), crRNA (0.4 μL, final concentration 400 nM), ssDNA FQ reporter gene (1 μL, final concentration 1 μM), Recombinant RNase Inhibitor (0.4 μL), 10×Cas12a Buffer (1 μL), RT-ERA product (1-2 μL) and DEPC water (supplementing the system to 10 μL); the reaction conditions of the CRISPR-Cas12a detection system were 37 ° C for 20 min; the reaction was performed by QuantStudio TM 5. Real-time fluorescence quantitative PCR instrument detection to obtain the fluorescence values of different primer combinations.

[0057] The results are as follows Figure 2As shown, first, the downstream primer R3 was selected to screen the amplification efficiency of the upstream primers F1-F6, and the results showed that the upstream primer F6 had the highest amplification efficiency; secondly, the upstream primer F6 was anchored to screen the amplification efficiency of the downstream primers R1-R8, and the results showed that the primer pair of upstream primer F6 (nucleotide sequence shown in SEQ ID NO.8) and downstream primer R3 (nucleotide sequence shown in SEQ ID NO.11) had the highest amplification efficiency.

[0058] Table 2

[0059] Prime name Prime sequence F1 5’-GTTAAAATGCAATGGTCGCAATYACCGTGC-3’ SEQ ID NO.3 F2 5’-GCAATGGTCGCAATYACCGTGCAAGGTGC-3’ SEQ ID NO.4 F3 5’-ATGCAATGGTCGCAATYACCGTGCAAGGTGC-3’ SEQ ID NO.5 F4 5’-GTTAAAATGCAATGGTCGCAATYACCGTG-3’ SEQ ID NO.6 F5 5’-GTTAAAATGCAATGGTCGCAATYACCGT-3’ SEQ ID NO.7 F6 5’-GTTAAAATGCAATGGTCGCAATYACCG-3’ SEQ ID NO.8 R1 5’-TTYYCTCTDAYRCGYCTKATATGDTCTG-3’ SEQ ID NO.9 R2 5’-TGYTTYYCTCTDAYRCGYCTKATATGDTCTG-3’ SEQ ID NO.10 R3 5’-TCCCARGTYGARTCAYGYARAACRGG-3’ SEQ ID NO.11 R4 5’-CYTCCCARGTYGARTCAYGYARAACRG-3’ SEQ ID NO.12 R5 5’-TCYTCCCARGTYGARTCAYGYARAACRGG-3’ SEQ ID NO.13 R6 5’-TTCYTCCCARGTYGARTCAYGYARAACRGG-3’ SEQ ID NO.14 R7 5’-CGYCTKATATGDTCTGAAAAYTTATARAAYTC-3’ SEQ ID NO.15 R8 5’-CTKATATGDTCTGAAAAYTTATARAAYTC-3’ SEQ ID NO.16

[0060] Note: Y represents the degenerate base C+T; D represents the degenerate base G+A+T; R represents the degenerate base A+G; K represents the degenerate base G+T.

[0061] (2) Sensitivity verification of the RT ERA / CRISPR-Cas12a detection system

[0062] In this example, the T7 in vitro transcription kit was used to transcribe the RNA template of the EHDV genome segment S1. The in vitro transcription volume was 20 μL, consisting of 10× Reaction Buffer (2 μL), 100 mM ATP / GTP / CTP / UTP (2 μL each), DNA template (2 μL, about 0.5 μg), T7 RNA Polymerase Mix (2 μL) and DEPC water (6 μL). The in vitro transcribed RNA product was then purified using the GeneJET RNA purification and concentration microextraction kit to obtain the EHDV S1 RNA template. In this example, the concentration of the detection was 1.7×10 4 , 1.7×10 3 , 1.7×10 2 , 1.7×10 1The EHDV S1 RNA template was prepared by adding 100 copies / μL of the RT-ERA amplification kit obtained in step (1) to react, and the obtained RT-ERA amplification product was used as a template for the CRISPR-Cas12a detection system; the CRISPR-Cas12a detection system had a reaction volume of 10 μL and consisted of Cas12a (75 ng), crRNA (0.4 μL, final concentration 400 nM), ssDNAFQ reporter gene or ssDNA biotin-labeled nucleic acid probe (1 μL, final concentration 1 μM), Recombinant RNase Inhibitor (0.4 μL), 10×Cas12a Buffer (1 μL), RT-ERA amplification product (1-2 μL) and DEPC water (replenish the system to 10 μL); CRISPR-Cas12a detection result determination: a. Perform CRISPR-Cas12a detection reaction in a real-time fluorescence quantitative PCR instrument, and set the fluorescence value to be read every 30 seconds; b. Observe under a blue light gel cutting instrument. After the CRISPR-Cas12a detection reaction is completed, take pictures under the blue light gel cutting instrument to record the brightness and color changes of the reaction tube. When a fluorescent signal appears in the reaction tube, the test result is positive. When no fluorescent signal appears in the reaction tube, the test result is negative. c. Use a lateral flow test strip. After the CRISPR-Cas12a detection reaction is completed, the result is judged by a lateral flow test strip. When the test line T line and the control line C line appear on the test strip at the same time, the test result is positive. When only the control line C line appears on the test strip, the test result is negative.

[0063] The results are as follows Figure 3 As shown, the RT ERA / CRISPR-Cas12a detection reaction was performed using the RNA of the EHDV genome segment S1 as a template. The detection limit based on the fluorescence signal was 1.7×10 1 copies / μL, and the lower limit of detection based on the lateral flow test strip was 1.7×10 2 The RT ERA / CRISPR-Cas12a detection system can determine the test results within 50 minutes, indicating that the epidemic hemorrhagic disease virus detection method provided by the present invention can achieve high-sensitivity, rapid and visual detection.

[0064] (3) Specificity verification of RT ERA / CRISPR-Cas12a detection

[0065] In this example, bluetongue virus (BTV), akapanax virus (AKAV), Chushan disease virus (CHUV), peste des petits ruminants virus (PPRV), goat pox virus (GPV), bovine respiratory syncytial virus (BRSV), bovine rotavirus (BRV), bovine parainfluenza virus type 3 (BPIV3) and bovine infectious rhinotracheitis virus (IBRV) were selected as the control group. The above viruses were donated by the Cattle and Sheep Infectious Diseases Research Innovation Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0066] According to the RT ERA / CRISPR-Cas12a detection system provided in this example, the above 9 control viruses and EHDV were specifically verified. The results are as follows Figure 4 As described above, except for the EHDV genomic RNA template, the other 9 control virus genomic RNAs all detected baseline fluorescence signals and negative test strip results, indicating that the visual epidemic hemorrhagic disease virus detection kit (RT-ERA / CRISPR-Cas12a system) provided by the present invention has high specificity for the detection of EHDV, and has no cross-reaction with other insect-borne viral infectious pathogens of ruminants BTV, AKAV, CHUV and common viral infectious pathogens of ruminants PPRV, GPV, BRSV, BRV, BPIV3 and IBRV.

[0067] (4) Feasibility verification of detecting different serotypes of EHDV

[0068] In this example, an RNA extraction kit was used to extract EHDV-5 and EHDV-7 genomic RNA as samples to be tested, and the S1 segments of EHDV-1, EHDV-2, EHDV-4, EHDV-6, EHDV-8, and EHDV-10 were synthesized. The RNA was transcribed by a T7 in vitro transcription kit as a sample to be tested, and RT-ERA amplification was performed by upstream primer F6 (nucleotide sequence shown in SEQ ID NO.8) and downstream primer R3 (nucleotide sequence shown in SEQ ID NO.11). The RT-ERA amplification product was used as a template for CRISPR-Cas12a detection reaction to verify the feasibility of the RT ERA / CRISPR-Cas12a detection system for detecting different serotypes of EHDV.

[0069] The results are as follows Figure 5As shown in the RT ERA / CRISPR-Cas12a detection reaction, when EHDV-5 and EHDV-7 genomic RNA, and EHDV-1, EHDV-2, EHDV-4, EHDV-6, EHDV-8 and EHDV-10 genomic segment S1 RNA were used as detection samples, high fluorescence signal values were detected, confirming that the visualized epidemic hemorrhagic disease virus detection method provided by the present invention is feasible for detecting different serotypes of EHDV.

[0070] Example 3: Preparation of a kit for visual detection of epidemic hemorrhagic disease virus

[0071] The kit described in this embodiment includes RT-EAR primers and crRNA; the forward primer nucleotide sequence of the RT-ERA primer is shown in SEQ ID NO.8, and the reverse primer nucleotide sequence is shown in SEQ ID NO.11; the crRNA comprises: crRNA1 with a nucleotide sequence shown in SEQ ID NO.1 and crRNA2 with a nucleotide sequence shown in SEQ ID NO.2;

[0072] The RT-ERA amplification system in the kit is: 4 μL of solvent, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of RNA template of the sample to be tested, 3.6 μL of DEPC water, and finally 0.4 μL of activator;

[0073] The kit also includes a CRISPR-Cas12a detection system, which consists of the following components: 75 ng of Cas12a, 0.4 μL of 10 μM crRNA, 1 μL of 10 μM ssDNA FQ reporter gene or 1 μL of ssDNA biotin-labeled nucleic acid probe, 0.4 μL of Recombinant RNase Inhibitor, 1 μL of 10×Cas12a Buffer, 1-2 μL of RT-ERA amplification product to be detected, and DEPC water to replenish the system to 10 μL.

[0074] Example 4: Application of a kit for visual detection of epidemic hemorrhagic disease virus

[0075] In this example, 54 blood and tissue samples were collected clinically. RNA was extracted from the blood and tissue samples using TRIzol reagent. Fluorescent RT-PCR and the kit for visual detection of epidemic hemorrhagic disease virus obtained in Example 3 (hereinafter referred to as the RT-ERA / CRISPR-Cas12a system) were used to detect epidemic hemorrhagic disease virus, and the test results were compared and analyzed.

[0076] In this example, the RT-ERA / CRISPR-Cas12a system was used to detect 54 samples to be tested, and the results were visualized using fluorescence signals and lateral flow test strips. The visual detection results of the lateral flow test strips were as follows: Figure 6 As shown, 23 were positive and 31 were negative, with a positive rate of 42.59%; secondly, the laboratory fluorescence RT-PCR detection method was used to detect 54 samples to be tested, and the detection results of RT-ERA / CRISPR-Cas12a fluorescence signals were compared with the laboratory fluorescence RT-PCR detection results. The results are shown as follows Figures 7-8 As shown, the detection results based on the fluorescence signal of the RT-ERA / CRISPR-Cas12a system and the visual detection results based on the lateral flow test strip are consistent with the laboratory fluorescence RT-PCR detection results, indicating that the kit for visual detection of epidemic hemorrhagic disease virus provided by the present invention can be used as a rapid visual detection tool for EHDV nucleic acid.

[0077] In this example, in order to verify the feasibility of the kit for visual detection of epidemic hemorrhagic disease virus for field testing, the 54 blood and tissue samples collected above were subjected to nucleic acid release, 50 μL of the tissue grinding solution supernatant or 50 μL of the blood was added to 450 μL of nucleic acid release agent, mixed, incubated at 95°C for 5 minutes, and centrifuged for 2 minutes. The supernatant was then taken for detection by the RT-ERA / CRISPR-Cas12a system, and the results were read by fluorescence signal to evaluate the field detection capability.

[0078] The results are as follows Figure 9 As shown, a total of 54 samples were rapidly tested using a nucleic acid releaser and the RT ERA / CRISPR-Cas12a system, with a fluorescence visualization positivity rate of 40.74% (22 / 54), which was highly consistent with the laboratory fluorescence RT-PCR detection positivity rate of 42.59% (23 / 54). This indicates that the kit for visual detection of epidemic hemorrhagic disease virus provided by the present invention can obtain visual detection results with a high consistency with the laboratory fluorescence RT-PCR detection results without relying on precision instruments, and is suitable for on-site detection work at the grassroots level.

[0079] The contents not described in detail in the present specification are well-known technologies to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A kit for visually detecting epidemic hemorrhagic disease virus, characterized in that: The kit includes an RT-ERA amplification system and a CRISPR-Cas12a detection system; The RT-ERA amplification system includes RT-ERA primers, the forward primer nucleotide sequence of the RT-ERA primers is shown in SEQ ID NO.8, and the reverse primer nucleotide sequence is shown in SEQ ID NO.11; The CRISPR-Cas12a detection system includes crRNA, Cas12a, ssDNA FQ reporter gene or ssDNA biotin-labeled nucleic acid probe, recombinant RNase inhibitor, and Cas12a Buffer; the crRNA nucleotide sequence is shown in SEQ ID NO.

1.

2. The kit according to claim 1, wherein The RT-ERA primer and crRNA are based on the EHDV genome segment S1 conserved sequence fragment as a template.

3. The kit according to claim 1, wherein The CRISPR-Cas12a detection system includes: 0.4 μL of 10 μM crRNA, 75 ng of Cas12a, 10 μM ssDNA FQ reporter gene or 1 μL of ssDNA biotin-labeled nucleic acid probe, 0.4 μL of recombinant RNase inhibitor, and 1 μL of 10×Cas12a Buffer.

4. A method for using the kit according to any one of claims 1 to 3 for non-diagnostic purposes, characterized in that: The method of use comprises the following steps: subjecting the sample RNA to be tested to RT-ERA isothermal amplification, reacting at 40°C for 20 minutes to obtain an RT-ERA amplification product; subjecting the RT-ERA amplification product to a CRISPR-Cas12a detection reaction, reacting at 37°C for 20 minutes to obtain a reactant, and then performing result detection on the reactant.

5. The method of use according to claim 4, characterized in that: The method for detecting the result in the method of use includes: real-time fluorescence quantitative PCR analysis, blue light gel cutting instrument analysis or lateral flow test strip analysis.

6. The method of use according to claim 5, wherein: The detection steps of the real-time fluorescence quantitative PCR analysis are as follows: performing a CRISPR-Cas12a detection reaction in a real-time fluorescence quantitative PCR instrument, setting the fluorescence value to be read once every 30 seconds, and performing result detection according to the read fluorescence value.

7. The method of use according to claim 5, characterized in that: The detection steps of the blue light gel cutting instrument analysis are as follows: the reactant obtained after the CRISPR-Cas12a detection reaction is placed under a blue light gel cutting instrument for observation, and the brightness and color changes of the reaction tube are recorded by taking pictures. When the reaction tube shows a fluorescent signal under the blue light gel cutting instrument, it indicates that the test result is positive; when the reaction tube does not show a fluorescent signal under the blue light gel cutting instrument, it indicates that the test result is negative.

8. The method of use according to claim 5, characterized in that: The detection steps of the lateral flow test strip analysis are as follows: the reactant obtained after the CRISPR-Cas12a detection reaction is placed on the lateral flow test strip for interpretation of the test results. When the test line T and the control line C appear on the test strip at the same time, it indicates that the test result is positive; when only the control line C appears on the test strip, it indicates that the test result is negative.

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