A CRISPR-Cas13a-based Bluetongue virus-specific crRNA and its related kit and detection method

Abstract

The present invention relates to a CRISPR-Cas13a-based bluetongue virus-specific crRNA and related kits and detection methods, belonging to the field of virus detection technology. In order to solve the technical problems of existing BTV detection methods such as long time consumption, complicated operation, and inability to achieve on-site real-time detection, the present invention obtains a specific crRNA targeting the BTV genome segment S1 through sequence alignment and screening, and finds that it can detect BTV strains of different serotypes in the CRISPR reaction; designs an RT-ERA primer pair that can specifically amplify the target sequence, and the product can be used for downstream CRISPR reactions; the present invention combines RT-ERA technology with the CRISPR-Cas13a system to establish a rapid, highly sensitive, specific, and visually detectable BTV detection method, which can be used for laboratory detection of BTV and rapid on-site detection in farms.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a bluetongue virus-specific crRNA based on CRISPR-Cas13a and a related kit and detection method. Background Art

[0002] Bluetongue disease (BTD) is an infectious disease caused by the Bluetongue virus (BTV), a Culicoides insect vector that primarily infects domestic and wild ruminants. It is widely distributed worldwide. All ruminants (including sheep, goats, cattle, deer, and camels) are susceptible to BTV. The severity of the disease is influenced by multiple factors, including the viral serotype, susceptible animal species, breed, and age. Sheep are most susceptible. Symptoms include elevated body temperature, lethargy, anorexia, excessive salivation, edema of the upper lip, face, and neck, congestion and ulceration of the oral mucosa, and a cyanotic, bluish-purple tongue. Symptoms may include coronitis and laminitis. In severe cases, hoof shell loss can lead to mobility problems. Certain serotypes can cause miscarriage, stillbirth, and mummified fetuses in pregnant animals, resulting in significant economic losses. The World Organization for Animal Health (OIE) lists BTD as a notifiable animal disease, while my country classifies it as a Category II animal disease. BTV causes severe illness and even death in ruminants, resulting in significant losses to the livestock industry and threatening wildlife populations, including endangered species. Although my country has yet to experience a BTD outbreak, numerous strains of BTV, including various and unknown serotypes, have been isolated from ruminants and vector insects, posing a significant threat to the country's growing ruminant industry.

[0003] BTV belongs to the genus Orbivirus, family Reoviridae. Its virions are non-enveloped, approximately 80 nm in diameter, and exhibit icosahedral symmetry. Its genome consists of 10 segmented double-stranded RNA (dsRNA), encoding seven structural proteins (VP1-VP7) and five nonstructural proteins (NS1, NS2, NS3, NS3A, and NS4). The outer capsid of the virion is composed of VP2 and VP5, while the inner capsid consists of VP3 and VP7. VP1, VP4, and VP6 form the replicase complex within. VP2 induces specific neutralizing antibodies in the host, which determine BTV serotype. BTV has numerous serotypes. Currently, at least 29 different BTV serotypes (BTV-1 to BTV-29) have been isolated and identified worldwide, as well as several BTV isolates for which serotypes have yet to be definitively determined, tentatively designated BTV-30 to BTV-36. There is no cross-protection between serotypes.

[0004] BTV detection methods include virus isolation and identification, serological testing, and nucleic acid testing. Virus isolation and identification requires specialized laboratories and has a long testing cycle. Serological testing is currently a universally recognized testing method for epidemiological surveys, customs import and export quarantine, and livestock breeding, but it is complex and unsuitable for field use. RT-PCR and fluorescent RT-PCR are commonly used techniques for pathogen nucleic acid detection, but they rely on specialized equipment and have a long reaction time. Therefore, existing detection methods are unable to meet the needs of rapid field detection of BTV. Therefore, there is an urgent need for a new method suitable for field detection that is rapid, highly sensitive, and specific, and can produce visual results without relying on sophisticated laboratory instruments. Summary of the Invention

[0005] To address the technical problems of existing BTV detection methods, such as being time-consuming, requiring expensive instruments or specialized technicians, being complicated to operate, and being unable to achieve real-time on-site detection, the present invention combines reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) technology with the CRISPR-Cas13a system to establish a method for visual detection of BTV based on nucleic acid release, RT-ERA, and CRISPR-Cas13a.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a BTV genome segment S1-specific crRNA based on CRISPR-Cas13a, wherein the BTV genome segment S1-specific crRNA is a mixture of the crRNAs shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in equal proportions.

[0008] The second object of the present invention is to provide the use of the above-mentioned BTV genome segment S1-specific crRNA in the preparation of a kit for detecting BTV.

[0009] The third object of the present invention is to provide a CRISPR-Cas13a-based reagent for detecting different serotypes of BTV, wherein the reagent comprises the BTV genome segment S1-specific crRNA according to claim 1.

[0010] The fourth object of the present invention is to provide use of the above reagent in preparing a kit for detecting BTV.

[0011] The fifth object of the present invention is to provide a CRISPR-Cas13a-based kit for detecting different serotypes of BTV, wherein the kit comprises the above-mentioned BTV genome segment S1-specific crRNA.

[0012] In one embodiment of the present invention, the kit further contains any one or more of Cas13a, ssRNA reporter system, recombinant RNase inhibitor, 10×Cas13aBuffer, ATP, GTP, CTP, UTP, T7 RNA Polymerase Mix, 10×T7 Reaction Buffer and BTV genome segment S1 isothermal amplification reagent.

[0013] In one embodiment of the present invention, the ssRNA reporter system is a ssRNAFQ reporter gene or a ssRNA biotin-labeled nucleic acid probe.

[0014] In one embodiment of the present invention, the BTV genome segment S1 isothermal amplification reagent includes an isothermal amplification primer pair, the upstream primer of the isothermal amplification primer pair is the nucleotide sequence shown in SEQ ID NO.11, and the downstream primer is the nucleotide sequence shown in SEQ ID NO.20.

[0015] A sixth object of the present invention is to provide the use of the above kit in the visualization detection of BTV for non-disease diagnosis purposes.

[0016] A seventh object of the present invention is to provide a method for visually detecting BTV for non-disease diagnosis purposes, wherein the method uses the above-mentioned kit for detection and comprises the following steps:

[0017] S1. Take a sample to be tested, and use a nucleic acid extraction reagent to extract nucleic acid in the sample, or use a nucleic acid release agent to release nucleic acid in the sample;

[0018] S2, adding the nucleic acid obtained in S1 and the above-mentioned isothermal amplification primer pair into the RT-ERA system for amplification to obtain a specific product;

[0019] S3, adding the specific product obtained in S2 to the CRISPR-Cas13a detection system to identify and cut the specific product;

[0020] S4. Use a blue light gel cutting instrument or lateral flow test strips to directly detect and determine whether BTV-specific nucleic acid is present in the sample.

[0021] In one embodiment of the present invention, the specific step of S1 is to mix the sample to be tested and the nucleic acid releasing agent in a volume ratio of 1:9, and incubate at 95° C. for 5 minutes.

[0022] In one embodiment of the present invention, the RT-ERA system described in S2 is 10 μL, consisting of a solubilizer (4 μL), a forward primer F2 (0.5 μL, a final concentration of 500 nM), a reverse primer R5 (0.5 μL, a final concentration of 500 nM), an RNA template (1 μL), DEPC water (3.6 μL) and an activator (0.4 μL).

[0023] In one embodiment of the present invention, the RT-ERA reaction conditions in S2 are 40° C. and 20 min.

[0024] In one embodiment of the present invention, the CRISPR-Cas13a detection system described in S3 is a 10 μL reaction system, consisting of Cas13a (75 ng), crRNA1, crRNA2 and crRNA3 (0.33 μL each, a final concentration of 330 nM), ssRNA FQ reporter gene or ssRNA biotin-labeled nucleic acid probe (1 μL, a final concentration of 1 μM), recombinant RNase inhibitor (Recombinant RNase Inhibitor, RRI) (0.4 μL), 10×Cas13a Buffer (1 μL), ATP / GTP / CTP / UTP (0.5 μL each, a final concentration of 5 mM), T7 RNA Polymerase Mix (1 μL), 10×T7 Reaction Buffer (1 μL), RT-ERA amplification product (product obtained in S2) (1-2 μL) and DEPC water (supplementing the system to 10 μL).

[0025] In one embodiment of the present invention, the reaction conditions for the CRISPR-Cas13a detection in S3 are 37° C. and 20 min.

[0026] Beneficial effects of the present invention:

[0027] (1) The principle of the BTV detection method based on the RT-ERA / CRISPR-Cas13a system is that after Cas13a forms a complex with crRNA to recognize the target sequence, it activates the incidental cleavage activity, cuts the ssRNA reporter probe in the system, and releases a fluorescent signal. This method can detect nucleic acids of different serotypes of BTV strains and has the advantages of strong specificity, high sensitivity, fast detection speed, visualization, and easy on-site application. It can be used for clinical sample analysis, on-site testing, daily monitoring, etc. in farms, providing a visualization technology platform for grassroots testing.

[0028] (2) The BTV nucleic acid detection method established based on nucleic acid extraction combined with the RT-ERA / CRISPR-Cas13a system can complete efficient and visualized laboratory testing. The results showed that the fluorescence signal positivity rate was 16.35% (34 / 208), the fluorescence visualization positivity rate was 16.35% (34 / 208), and the test strip visualization detection positivity rate was 14.90% (31 / 208). All of them were highly consistent with the detection results of the fluorescence RT-PCR detection method in the national standard GB / T18636-2017 bluetongue disease diagnostic technology of 16.35% (34 / 208), and are suitable for laboratory testing.

[0029] (3) The BTV nucleic acid detection method based on nucleic acid release combined with RT-ERA / CRISPR-Cas13a system can complete efficient, convenient and visual on-site detection. The results showed that the fluorescence visualization positive rate was 15.38% (32 / 208), which was highly consistent with the detection result of 16.35% (34 / 208) of the fluorescence RT-PCR detection method in the national standard GB / T 18636-2017 bluetongue disease diagnostic technology. This method does not rely on precision instruments and can obtain visual detection results within 1 hour without the need for professional technicians. It is suitable for on-site detection work at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a result diagram of the detection efficiency of the CRISPR-Cas13a system mediated by crRNA combinations targeting different segments using a real-time fluorescence quantitative PCR instrument; Figure 1 A in the figure is the detection efficiency result diagram of crRNA combination targeting different segments against BTV-1 strains, Figure 1 Figure B shows the detection efficiency of crRNA combinations targeting different segments against BTV-15 strains. Figure 1 Figure C shows the detection efficiency of crRNA combinations targeting different segments against BTV-16 strains. Figure 1 D in the figure is the detection efficiency result diagram of crRNA combinations targeting different segments against BTV-20 strains;

[0031] Figure 2 is the screening result diagram of RT-ERA primers; wherein, Figure 2 A in the figure is the screening result of RT-ERA upstream primer. Figure 2 Figure B is the screening result of RT-ERA downstream primers;

[0032] Figure 3 This is the sensitivity verification result diagram of the RT-ERA / CRISPR-Cas13a detection method; Figure 3A in the figure is a result graph showing the sensitivity of the RT-ERA / CRISPR-Cas13a detection method using fluorescence readings. Figure 3 Figure B is a result diagram showing the sensitivity of the RT-ERA / CRISPR-Cas13a detection method using fluorescence signals. Figure 3 C in the figure is a result graph showing the sensitivity of the RT-ERA / CRISPR-Cas13a detection method using a test strip;

[0033] Figure 4 This is the specificity verification result diagram of the RT-ERA / CRISPR-Cas13a detection method; Figure 4 A in the figure is a result diagram showing the specificity of the RT-ERA / CRISPR-Cas13a detection method using fluorescence readings. Figure 4 Figure B is a result diagram showing the specificity of the RT-ERA / CRISPR-Cas13a detection method using fluorescence signals. Figure 4 C in the figure is a result diagram of the specificity of the RT-ERA / CRISPR-Cas13a detection method using a test strip;

[0034] Figure 5 The fluorescence reading results of RT-ERA / Cas13a detection of different serotypes of BTV strains genomic RNA and synthetic in vitro transcribed RNA containing base mismatches S1 sequence; Figure 5 A in the figure is the fluorescence reading result of RT-ERA / Cas13a detecting genomic RNA of different serotypes of BTV strains. Figure 5 Figure B shows the fluorescence reading result of RT-ERA / Cas13a detecting the in vitro transcribed RNA of the synthetic S1 sequence containing base mismatches;

[0035] Figure 6 This is a statistical graph showing the results of detecting RNA extracted from clinical samples using RT-ERA / Cas13a fluorescence signal and fluorescent RT-PCR detection methods respectively;

[0036] Figure 7 This is a statistical chart showing the results of testing RNA extracted from clinical samples using the RT-ERA / Cas13a test strip method and the fluorescent RT-PCR detection method;

[0037] Figure 8 This figure shows the results of detecting RNA extracted from clinical samples using RT-ERA / Cas13a fluorescence signals and the test strip method;

[0038] Figure 9This is a statistical graph of the results of using nucleic acid release, RT-ERA / Cas13a fluorescence signal detection of clinical samples and using fluorescent RT-PCR to detect RNA extracted from clinical samples. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific examples and accompanying drawings. The embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements are within the scope of protection of the present invention. The process, conditions, experimental methods and reagents for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and conventional products in the market, and the present invention does not particularly limit the content.

[0040] HSDNA Polymerase and Recombinant RNase Inhibitor were purchased from Bio-Technology Co., Ltd.; GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; T7 High Yield RNA Transcription Kit was purchased from Nanjing Novozymes Biotech Co., Ltd.; GenCRISPR TM Cas13a (C2c2) Nuclease was purchased from GenScript Biotech Co., Ltd.; RT-based nucleic acid amplification reagent (ERA method), lateral flow test strips (CRISPR), and nucleic acid release agents were purchased from Suzhou Xinda Gene Technology Co., Ltd.; crRNA was synthesized by GenScript Biotech Co., Ltd.; DNA sequences were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.; inactivated BTV strains of different serotypes, epidemic hemorrhagic disease virus (EHDV), Akabane virus (AKAV), Zhongshan 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 donated by the Cattle and Sheep Infectious Diseases Innovation Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0041] Example 1: Screening of crRNA and construction of CRISPR-Cas13a detection system

[0042] 1. crRNA design

[0043] To ensure the universality and specificity of the BTV detection method, sequences of BTV strains of different serotypes were screened from GenBank as references. Sequence alignment initially selected segments S1, S8, S9, and S10 as detection targets. CrRNA combinations were designed based on the protospacer flanking sequence (PFS) sites. CrRNA combinations targeting different genomic segments consisted of two or three sequences: crRNA1 and crRNA2, or crRNA1, crRNA2, and crRNA3 (see Table 1).

[0044] Table 1 crRNA sequence information designed for different segments of the BTV genome

[0045]

[0046] 2. Construction of CRISPR-Cas13a detection system

[0047] The CRISPR-Cas13a detection system is a 10 μL reaction system consisting of Cas13a (75 ng), a combination of crRNA1 and crRNA2 (0.33 μL each, a final concentration of 330 nM), or a combination of crRNA1, crRNA2 and crRNA3 (0.33 μL each, a final concentration of 330 nM), ssRNAFQ reporter gene or ssRNA biotin-labeled nucleic acid probe (1 μL, a final concentration of 1 μM), recombinant RNase inhibitor (RRI) (0.4 μL), 10×Cas13a Buffer (1 μL), RNA template (1-2 μL) and DEPC water (replenish the system to 10 μL). The reaction temperature for CRISPR-Cas13a detection is 37°C, and the reaction time is 20 minutes. The test results can be read in a real-time fluorescence quantitative PCR instrument and recorded every 30 seconds. After the reaction is completed, the brightness and color changes of the reaction tube can be recorded by taking pictures under a blue light gel cutting instrument. After the reaction is completed, the results can also be interpreted using lateral flow test strips.

[0048] 3. Screening of crRNA

[0049] To evaluate the efficiency of the CRISPR-Cas13a system mediated by crRNA combinations targeting different segments, different crRNA sequence combinations targeting BTV genome segments S1, S8, S9, and S10 were incubated with the extracted genomic RNA of BTV-1, BTV-15, BTV-16, and BTV-20 strains for detection. The efficiency of different crRNA combinations was analyzed by real-time fluorescence quantitative PCR and the screening was completed for subsequent experiments.

[0050] like Figure 1 As shown in Figure 2, in crRNA screening, the cleavage results for BTV-1, BTV-15, BTV-16, and BTV-20 genomic segments S1, S8, S9, and S10 showed that the crRNA combination targeting genomic segment S1 had the highest cleavage efficiency, while the negative control group showed no reaction. Therefore, this combination was selected as the crRNA for downstream experiments.

[0051] Example 2: Establishment of RT-ERA / CRISPR-Cas13a detection system

[0052] 1. Design of isothermal amplification primers

[0053] According to the RT-ERA primer design principles, 6 upstream primers and 5 downstream primers were designed for the S1 segment of different BTV strains, and they were cross-paired and screened. The RT-ERA amplification reaction system was 10 μL, consisting of 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), a BTVS1 RNA template (1 μL, 10 3 A total of 100 copies of the PCR product (100 μL) and DEPC water (3.6 μL) were added, followed by the addition of an activator (0.4 μL). The RT-ERA amplification reaction was incubated at 40°C for 20 min. 1 μL of the RT-ERA amplification product was aspirated and used in the CRISPR-Cas13a detection system. The primer pair with the highest detection efficiency was determined based on the fluorescence value (same as in Example 1).

[0054] Table 2 RT-ERA isothermal amplification primers designed for BTV genome segment S1

[0055]

[0056] First, the downstream primer R3 was selected to screen the upstream primers F1-F6. The results showed that the upstream primer F2 had the highest amplification efficiency. Then, the upstream primer F2 was anchored and the downstream primers R1-R5 were screened. The results showed that the primer pair F2 and R5 had the highest amplification efficiency (see Figure 2 ).

[0057] 2. Sensitivity verification of RT-ERA / CRISPR-Cas13a detection method

[0058] In sensitivity validation, an RNA template was generated by transcription using a T7 in vitro transcription kit. Specifically, a nucleotide sequence of the BTV genomic segment S1 containing a T7 promoter sequence was synthesized and used as a DNA template. The in vitro transcription system consisted of 20 μL of 10× Reaction Buffer (2 μL), 2 μL each of ATP / GTP / CTP / UTP, DNA template (2 μL, approximately 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 Kit and used as the RNA template for subsequent reactions.

[0059] The detection concentrations of CRISPR-Cas13a combined with RT-ERA (RT-ERA / CRISPR-Cas13a detection method) were 2×10 3 , 2×10 2 , 2×10 1 , 2×10 0 Copies / μL of BTV S1 RNA template. The reaction was carried out using the RT-ERA amplification reaction system (same as step 1 in Example 2, and the amplification primers F2 and R5 were selected). The RT-ERA amplification product was used for the CRISPR-Cas13a detection reaction. The CRISPR-Cas13a detection system was a 10 μL reaction system consisting of Cas13a (75 ng), crRNA1, crRNA2, and crRNA3 (0.33 μL each, a final concentration of 330 nM), ssRNAFQ reporter gene or ssRNA biotin-labeled nucleic acid probe (1 μL, a final concentration of 1 μM), recombinant RNase inhibitor (RRI) (0.4 μL), 10 × Cas13a Buffer (1 μL), ATP / GTP / CTP / UTP (0.5 μL each, a final concentration of 5 mM), T7 RNA Polymerase Mix (1 μL), 10 × T7 Reaction Buffer (1 μL), RT-ERA amplification product (1-2 μL) and DEPC water (replenish the system to 10 μL), the reaction temperature is 37°C, the reaction time is 20 min, and the sensitivity of BTV detection is determined based on its fluorescence reading, fluorescence signal and test strip.

[0060] Sensitivity validation results showed that the fluorescence reading, fluorescence signal and detection limit of BTV genomic RNA on the test strip were 2×10 1The detection results can be identified within 20 minutes, indicating that the RT-ERA / CRISPR-Cas13a detection method can achieve high-sensitivity and rapid visual detection (see Figure 3 ).

[0061] 3. Specificity verification of RT-ERA / CRISPR-Cas13a detection method

[0062] In the specificity verification, EHDV, AKAV and CHUV, pathogens of insect-borne infectious diseases of cattle and sheep, as well as PPRV, GPV, BRSV, BRV, BPIV3 and IBRV, other common infectious pathogens of cattle and sheep, were selected as controls. The RT-ERA / CRISPR-Cas13a system was used for detection reactions, and the specificity of the detection method was determined by fluorescence readings, fluorescence signals and test strips.

[0063] The specificity validation results showed that, except for the BTV genomic RNA template, the baseline fluorescence signal and negative test strip results were detected for other viral genomic RNAs, indicating that the RT-ERA / CRISPR-Cas13a detection method has high specificity (see Figure 4 ).

[0064] 4. Detection of different serotypes of BTV

[0065] BTV strains of different serotypes (BTV-1 / -2 / -4 / -7 / -9 / -12 / -15 / -16 / -20 / -21) were selected as test samples, viral RNA was extracted using an RNA extraction kit, and RT-ERA / CRISPR-Cas13a detection was performed using the above-established detection method; at the same time, the S1 sequences of the genomic segments of BTV strains of different serotypes (BTV-6 / -14 / -25 / -28 / -29 / -33, no corresponding viruses were available) that had individual base mismatches with the crRNA1, crRNA2, or crRNA3 sequences were synthesized, and RNA was obtained by in vitro transcription as a test sample for RT-ERA / CRISPR-Cas13a detection.

[0066] The detection capability of RT-ERA / CRISPR-Cas13a was evaluated using genomic RNA of different serotypes of BTV strains and synthetic in vitro transcribed RNA containing base mismatches as test samples. The results showed that both had high fluorescence values, confirming that this method can be used to detect different serotypes of BTV (see Figure 5 ).

[0067] Example 3: A method for visual detection of BTV based on RT-ERA and CRISPR-Cas13a

[0068] 1. A method for visual detection of BTV based on nucleic acid release, RT-ERA, and CRISPR-Cas13a

[0069] Nucleic acid release was combined with the CRISPR-Cas13a detection system obtained in Example 1 and the RT-ERA system obtained in Example 2 to obtain a method for visual detection of BTV based on nucleic acid release, RT-ERA, and CRISPR-Cas13a, which specifically includes the following steps:

[0070] S1. Take the sample to be tested and use a nucleic acid releaser to release the nucleic acid in the sample. The volume ratio of the sample to the nucleic acid releaser is 1:9. After mixing the sample and the nucleic acid releaser according to the volume ratio, incubate at 95°C for 5 minutes to fully release the nucleic acid.

[0071] S2. Add the nucleic acid obtained in S1 and the isothermal amplification primer pair to the RT-ERA amplification system for amplification to obtain a specific amplification product; the RT-ERA amplification system is 10 μL, consisting of a solvent (4 μL), a forward primer F2 (0.5 μL, a final concentration of 500 nM), a reverse primer R5 (0.5 μL, a final concentration of 500 nM), an RNA template (1 μL) and DEPC water (replenish the system to 10 μL), and then add an activator (0.4 μL). The conditions of the RT-ERA amplification reaction are 40°C and the reaction time is 20 min.

[0072] S3. The specific amplification product obtained in S2 was added to the CRISPR-Cas13a detection system to identify and cut the specific product. The CRISPR-Cas13a detection system was a 10 μL reaction system, consisting of Cas13a (75 ng), crRNA1, crRNA2 and crRNA3 (0.33 μL each, with a final concentration of 330 nM), ssRNAFQ reporter gene or ssRNA biotin-labeled nucleic acid probe (1 μL, with a final concentration of 1 μM), recombinant RNase inhibitor (RRI) (0.4 μL), 10×Cas13a Buffer (1 μL), ATP / GTP / CTP / UTP (0.5 μL each, with a final concentration of 5 mM), T7 RNA Polymerase Mix (1 μL), 10×T7 Reaction Buffer (1 μL), RT-ERA amplification product (product obtained by S2) (1-2 μL) and DEPC water (replenish the system to 10 μL); the detection conditions are 37°C and the reaction time is 20 min.

[0073] S4. Use a blue light gel cutting instrument or lateral flow test strips to directly determine whether BTV-specific nucleic acid is present in the sample.

[0074] 2. Laboratory visualization of clinical samples using RT-ERA / CRISPR-Cas13a detection method

[0075] To verify the feasibility of the RT-ERA and CRISPR-Cas13a-based detection method proposed in Example 2 for clinical sample detection, 208 blood and tissue samples were collected, and RNA was extracted using TRIzol reagent. Then, the fluorescent RT-PCR detection method (GB / T 18636-2017 Bluetongue Diagnosis Technology) and the RT-ERA / CRISPR-Cas13a detection method were used simultaneously, and the test results were compared to evaluate the detection capability.

[0076] 208 samples were tested using both fluorescent RT-PCR and RT-ERA / CRISPR-Cas13a systems. The results showed that the detection results of the RT-ERA / CRISPR-Cas13a fluorescent signal were consistent with those of the fluorescent RT-PCR detection method. 34 samples were positive and 174 samples were negative, with a positive rate of 16.35%. This indicates that the efficiency of the established RT-ERA / CRISPR-Cas13a detection method is consistent with that of the fluorescent RT-PCR detection method (see Figure 6 The positive rate of RT-ERA / CRISPR-Cas13a test strip visualization detection was 14.90% (31 / 208), and it also had a high consistency rate with the fluorescent RT-PCR detection method (see Figure 7 and Figure 8 ), indicating that this method can be used for rapid visual detection of BTV nucleic acid.

[0077] 3. On-site detection using nucleic acid release and RT-ERA / CRISPR-Cas13a detection methods

[0078] To validate the feasibility of the nucleic acid release, RT-ERA / CRISPR-Cas13a-based method for field testing, 208 blood and tissue samples were tested. 50 μL of tissue abrasive supernatant or 50 μL of blood was added to 450 μL of nucleic acid release reagent, mixed, incubated at 95°C for 5 minutes, and centrifuged. The supernatant was then tested with the RT-ERA / CRISPR-Cas13a system. The results were read as fluorescence signals to assess the field detection capability.

[0079] A total of 208 samples were rapidly tested using the nucleic acid release and RT-ERA / CRISPR-Cas13a systems. The results showed that the fluorescence visualization positive rate was 15.38% (32 / 208), which was highly consistent with the laboratory fluorescence RT-PCR test result of 16.35% (34 / 208) (see Figure 9), indicating that the detection method based on nucleic acid release and RT-ERA / CRISPR-Cas13a can obtain visual detection results without relying on sophisticated instruments, and is suitable for on-site detection work at the grassroots level.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can 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 based on the definition of the claims.

Claims

1. A CRISPR-Cas13a-based BTV genome segment S1-specific crRNA, characterized in that The BTV genome segment S1-specific crRNA is a mixture of the crRNAs shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in equal proportions.

2. Use of the BTV genome segment S1-specific crRNA according to claim 1 in the preparation of a kit for detecting BTV.

3. A CRISPR-Cas13a-based reagent for detecting different serotypes of BTV, characterized in that: The reagent includes the BTV genome segment S1-specific crRNA according to claim 1.

4. Use of the reagent according to claim 3 in preparing a kit for detecting BTV.

5. A kit for detecting different serotypes of BTV based on CRISPR-Cas13a, characterized in that: The kit comprises the BTV genome segment S1-specific crRNA according to claim 1.

6. The kit according to claim 5, characterized in that The kit also contains Cas13a, an ssRNA reporter system, a recombinant RNase inhibitor, 10×Cas13a Buffer, ATP, GTP, CTP, UTP, T7 RNA Polymerase Mix, 10×T7 Reaction Buffer, and a BTV genome segment S1 isothermal amplification reagent.

7. The kit according to claim 6, characterized in that The BTV genome segment S1 isothermal amplification reagent includes an isothermal amplification primer pair, wherein the upstream primer of the isothermal amplification primer pair is the nucleotide sequence shown in SEQ ID NO.11, and the downstream primer is the nucleotide sequence shown in SEQ ID NO.

20.

8. Use of the kit according to any one of claims 5 to 7 in visual detection of BTV for purposes other than disease diagnosis.

9. A method for visually detecting BTV for non-disease diagnosis purposes, characterized in that: The detection is performed using the kit according to claim 7, comprising the following steps: S1. Take a sample to be tested, and use a nucleic acid extraction reagent to extract nucleic acid in the sample, or use a nucleic acid release agent to release nucleic acid in the sample; S2. adding the nucleic acid obtained in S1 and the isothermal amplification primer pair described in claim 7 to a reverse transcription-recombinase polymerase amplification system for amplification to obtain a specific product; S3. Adding the specific product obtained in S2 to the CRISPR-Cas13a detection system to identify and cut the specific product, wherein the CRISPR-Cas13a detection system includes the BTV genome segment S1-specific crRNA described in claim 7; S4. Use a blue light gel cutting instrument or lateral flow test strips to directly determine whether BTV-specific nucleic acid is present in the sample.

10. The method according to claim 9, characterized in that The specific steps of S1 are to mix the sample to be tested and the nucleic acid release agent in a volume ratio of 1:9 and incubate at 95°C for 5 min.

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