CRISPR-Cas13a-based bluetongue virus specific crRNA and related kit and detection method thereof

Abstract

The invention relates to a bluetongue virus specific crRNA based on CRISPR-Cas13a as well as a related kit and a detection method thereof, and belongs to the technical field of virus detection. In order to solve the technical problems that an existing BTV detection method is long in time consumption, complex in operation, incapable of achieving on-site real-time detection and the like, specific crRNA of a targeted BTV genome segment S1 is obtained through sequence comparison and screening, and it is found that the specific crRNA can detect BTV strains of different serotypes in CRISPR reaction; an RT-ERA primer pair capable of specifically amplifying a target sequence is designed, and a product can be used for downstream CRISPR reaction; according to the present invention, the RT-ERA technology and the CRISPR-Cas13a system are combined to establish the BTV detection method with characteristics of rapidness, high sensitivity, strong specificity and result visualization, and the detection method can be used for the laboratory detection of BTV and the rapid on-site detection of farms.

Description

Technical Field

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

[0002] Bluetongue disease (BTD) is an infectious disease caused by the bluetongue virus (BTV) and transmitted by Culicoides vector insects, mainly infecting domestic and wild ruminants. It is widely distributed worldwide. All ruminants (including sheep, goats, cattle, deer, camels, etc.) are susceptible to BTV. The severity of the disease after infection is affected by multiple factors such as the virus serotype, susceptible animal species, breed, and age. Sheep are the most susceptible, with symptoms such as fever, mental depression, anorexia, excessive salivation, edema of the upper lip, face, and neck, congestion and ulceration of the oral mucosa, cyanosis of the tongue, and may be accompanied by coronitis and laminitis. In severe cases, the hoof shell falls off, causing difficulty in movement. Infection with certain serotypes of the virus can cause miscarriage, stillbirth, and mummification in pregnant animals, causing serious economic losses. The World Organization for Animal Health lists BTD as a legally notifiable animal disease, and my country lists it as a Class II animal disease. BTV causes serious diseases and even death in ruminants, which not only brings great losses to the livestock farming industry, but also threatens wild animal populations, including endangered species. Although there has not been an outbreak of BTD in my country, many strains of BTV of different serotypes and unknown serotypes have been isolated from ruminants and vector insects in China, which poses a huge threat to my country's growing ruminant farming industry.

[0003] BTV belongs to the genus Orbivirus of the family Reoviridae. The virus particles are enveloped, about 80 nm in diameter, and icosahedral in symmetry. The genome consists of 10 segmented double-stranded RNAs (dsRNA), encoding 7 structural proteins (VP1-VP7) and 5 non-structural proteins (NS1, NS2, NS3, NS3A, and NS4). The outer capsid of the virus particle is composed of VP2 and VP5, the inner capsid is composed of VP3 and VP7, and VP1, VP4, and VP6 form a replicase complex inside. VP2 induces the host to produce specific neutralizing antibodies, which determines the serotype of BTV. There are many serotypes of BTV. At present, at least 29 different serotypes of BTV (BTV-1 to BTV-29) have been isolated and identified worldwide, as well as several BTV isolates whose serotypes have not yet been finally confirmed, preliminarily identified as BTV-30 to BTV-36. There is no cross-protection between different serotypes.

[0004] BTV detection methods include virus isolation and identification, serological testing, and nucleic acid testing. Among them, virus isolation and identification must be completed in a professional laboratory, and the detection cycle is long; serological testing is currently recognized by the world as a universal inspection method in epidemiological surveys, customs import and export quarantine, and animal husbandry, but the operation is complicated and not suitable for field applications; RT-PCR and fluorescent RT-PCR are commonly used pathogen nucleic acid detection technologies, but they rely on professional equipment and have a long reaction time. In summary, the current detection methods cannot meet the needs of rapid on-site detection of BTV. Therefore, it is urgent to establish a new method suitable for on-site detection that is fast, highly sensitive, and highly specific, and can obtain visual results without relying on laboratory precision instruments. Summary of the invention

[0005] In order to solve the technical problems of existing BTV detection methods, such as being time-consuming, requiring expensive instruments or professional technicians, being complicated to operate, and being unable to achieve on-site real-time detection, the present invention combines reverse transcription-enzymatic recombinant 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 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 reagent for detecting different serotypes of BTV based on CRISPR-Cas13a, 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 the use of the above reagent in preparing a kit for detecting BTV.

[0011] A fifth object of the present invention is to provide a kit for detecting different serotypes of BTV based on CRISPR-Cas13a, 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 RNAPolymerase Mix, 10×T7ReactionBuffer 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 comprises an isothermal amplification primer pair, the upstream primer of the isothermal amplification primer pair is a nucleotide sequence as shown in SEQ ID NO.11, and the downstream primer is a nucleotide sequence as shown in SEQ ID NO.20.

[0015] The sixth object of the present invention is to provide the use of the above kit in BTV visualization detection 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 isothermal amplification primer pair mentioned above 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 a lateral flow test strip to directly detect and determine whether BTV-specific nucleic acid exists in the sample to be tested by naked eye visualization.

[0021] In one embodiment of the present invention, the specific step of S1 is to mix the sample to be tested with 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 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), 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 the reaction time is 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, with a final concentration of 330 nM), ssRNA FQ reporter gene or ssRNA biotin-labeled nucleic acid probe (1 μL, with 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, 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 in S2) (1-2 μL) and DEPC water (replenishing the system to 10 μL).

[0025] In one embodiment of the present invention, the reaction conditions of the CRISPR-Cas13a detection in S3 are 37° C., 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 detection, daily monitoring, etc. in farms, providing a visualization technology platform for grassroots detection.

[0028] (2) The BTV nucleic acid detection method 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 in high agreement 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 were 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 visualized on-site detection. The results showed that the fluorescence visualization positive rate was 15.38% (32 / 208), which was 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 diagnosis technology. This method does not rely on precision instruments and does not require professional technicians. Visual detection results can be obtained within 1 hour, which 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 combinations targeting different segments against BTV-1 strains. Figure 1 B in the figure is the detection efficiency result diagram of crRNA combinations targeting different segments against BTV-15 strains. Figure 1 C in the figure is the detection efficiency result diagram 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; among them, Figure 2 A in the figure is the screening result of RT-ERA upstream primer. Figure 2 B in the figure is the screening result diagram of RT-ERA downstream primers;

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

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

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

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

[0036] Figure 7 This is a statistical chart of 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 is a graph showing the results of using RT-ERA / Cas13a fluorescence signal and the test strip method to detect RNA extracted from clinical samples;

[0038] Fig. 9This is a statistical chart 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 is further described in detail below in conjunction with specific examples and accompanying drawings, and the embodiments are intended to illustrate the present invention only, rather than to limit the scope of the present 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 protection scope 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 in the art, and the present invention does not particularly limit the contents.

[0040] HSDNA Polymerase and Recombinant RNase Inhibitor were purchased from Baoriwu Biotechnology Co., Ltd.; GeneJET Gel Recovery Kit was purchased from Thermo Fisher Scientific; T7 High Yield RNA Transcription Kit was purchased from Nanjing Novozyme Biotechnology 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 releasers 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), akapan 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] In order to ensure the universality and specificity of the BTV detection method, the sequences of BTV strains of different serotypes were screened from GenBank as references. After sequence alignment, the S1, S8, S9, and S10 segments were preliminarily selected as detection targets, and crRNA combinations were designed according to the protospacer flanking sequence (PFS). The crRNA combinations targeting different segments of the genome contained 2 or 3 sequences, namely 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 (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 of 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 judged by lateral flow test strips.

[0048] 3. Screening of crRNA

[0049] In order 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 the figure, in the crRNA screening, the cleavage results for the genome segments S1, S8, S9, and S10 of BTV-1, BTV-15, BTV-16, and BTV-20 showed that the crRNA combination targeting the genome segment S1 had the highest cleavage efficiency, and the negative control group had no reaction. Therefore, this combination was selected as 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 principle, 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 The reaction conditions for RT-ERA amplification were incubation at 40°C for 20 min. 1 μL of RT-ERA amplification product was taken for CRISPR-Cas13a detection system, and the primer pair with the highest detection efficiency was determined according to 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 the sensitivity verification, the T7 in vitro transcription kit was used to transcribe the RNA template. Specifically, the nucleotide sequence of the BTV genome segment S1 containing the T7 promoter sequence was synthesized and used as a DNA template to transcribe the RNA template using the T7 in vitro transcription kit. The in vitro transcription system was 20 μL, consisting of 10× Reaction Buffer (2 μL), 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 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 1000 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, with amplification primers F2 and R5), and 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, 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 (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 (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] The sensitivity validation results showed that the fluorescence reading, fluorescence signal and BTV genomic RNA detection limit of 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 rapid visualization 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, and other common infectious pathogens of cattle and sheep PPRV, GPV, BRSV, BRV, BPIV3 and IBRV were selected as controls. The RT-ERA / CRISPR-Cas13a system was used for detection reaction, 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 genomic segment S1 sequence of different serotypes of BTV strains (BTV-6 / -14 / -25 / -28 / -29 / -33, no corresponding virus available) that had individual base mismatches with the crRNA1, crRNA2 or crRNA3 sequences was synthesized, and RNA was obtained by in vitro transcription as a test sample for RT-ERA / CRISPR-Cas13a detection.

[0066] The detection ability of RT-ERA / CRISPR-Cas13a was evaluated using genomic RNA of BTV strains of different serotypes and synthetic in vitro transcribed RNA containing base mismatches in the S1 sequence 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] The 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 the 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 an activator (0.4 μL) is added. The conditions of the RT-ERA amplification reaction are 40°C, and the reaction time is 20 min.

[0072] S3. Add the specific amplification product obtained in S2 to the CRISPR-Cas13a detection system to identify and cut the specific product; the CRISPR-Cas13a detection system is 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 (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 from 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 a lateral flow test strip to directly determine whether BTV-specific nucleic acid exists in the sample to be tested by naked eye visualization.

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

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

[0076] The fluorescent RT-PCR and RT-ERA / CRISPR-Cas13a systems were used to jointly detect 208 samples. The results showed that the detection results of the RT-ERA / CRISPR-Cas13a fluorescent signal were consistent with the results of the fluorescent RT-PCR detection method. 34 samples were positive and 174 samples were negative, with a positive rate of 16.35%. This shows 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] In order to verify the feasibility of the nucleic acid release, RT-ERA / CRISPR-Cas13a-based method for field detection, 208 blood and tissue samples were tested. 50 μL of tissue grinding liquid supernatant or 50 μL of blood was added to 450 μL of nucleic acid release agent, mixed, incubated at 95°C for 5 minutes, centrifuged and the supernatant was taken for RT-ERA / CRISPR-Cas13a system detection, and the results were read by fluorescence signal to evaluate 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 Fig. 9), indicating that the detection method based on nucleic acid release and RT-ERA / CRISPR-Cas13a can obtain visual detection results without relying on precision instruments, and is suitable for on-site detection work at the grassroots level.

[0080] Although the present invention has been disclosed as above in the form of preferred embodiments, it 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 BTV genome segment S1-specific crRNA based on CRISPR-Cas13a, characterized in that: The BTV genome segment S1-specific crRNA is a mixture of 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 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.

7. The kit according to claim 6, characterized in that The BTV genome segment S1 isothermal amplification reagent comprises an isothermal amplification primer pair, wherein the upstream primer of the isothermal amplification primer pair is a nucleotide sequence as shown in SEQ ID NO.11, and the downstream primer is a nucleotide sequence as 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 any one of claims 5 to 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; S4. Use a blue light gel cutting instrument or a lateral flow test strip to directly determine whether BTV-specific nucleic acid exists in the sample to be tested by naked eye visualization.

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

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