Bovine rotavirus detection reaction system, RAA, crRNA primers and detection methods and applications

Abstract

The present invention relates to the technical field of virus detection, and specifically discloses a bovine rotavirus detection reaction system, RAA, crRNA primers, and a detection method and application. The present invention provides a bovine rotavirus RAA-CRISPR / CAS reaction system, which comprises: bovine rotavirus RAA upstream and downstream primers and crRNA; the sequences of the bovine rotavirus RAA upstream and downstream primers are shown in SEQ ID NO.1-2, respectively, and the sequence of the crRNA is shown in SEQ ID NO.3. The reaction system can be used to perform on-site visual detection or fluorescence detection of bovine rotavirus genes without power supply, and the results can be quickly determined, with less detection equipment, low cost, high sensitivity and good specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a bovine rotavirus detection reaction system, RAA, crRNA primers, and a detection method and application. Background Art

[0002] Bovine rotavirus (BRV) is one of the common viral pathogens that cause calf diarrhea. After infection, calves will be characterized by mental fatigue, anorexia, vomiting, diarrhea, and dehydration. Rotavirus infection of calves and mixed infection with other intestinal pathogens will lead to aggravated diarrhea symptoms, and severe cases can cause death. It has become one of the main epidemic diseases affecting the healthy development of the cattle industry. At present, there is a lack of effective treatment for BRV infection, and there is little research on BRV vaccines, which makes the prevention and control of BRV more difficult. In addition, calf diarrhea is a common epidemic disease in clinical practice, and there are many pathogens that cause calf diarrhea, such as Escherichia coli, Salmonella, and Coronavirus. It is difficult to distinguish only from clinical symptoms and requires laboratory diagnosis. For this reason, it is particularly important to establish a one-tube nucleic acid detection method for BRV.

[0003] At present, the diagnostic methods for BRV mainly include traditional methods, such as virus isolation and identification, electron microscopy observation diagnosis; immunological detection technology, including enzyme-linked immunosorbent assay, immunochromatographic test strip detection technology; molecular biology technology, including RT-PCR, real-time fluorescence quantitative RT PCR, loop-mediated isothermal amplification technology, in situ hybridization technology, nanopore sequencing technology, etc.

[0004] However, the isolation and culture of the virus is mainly through the inoculation of MA 104 cells, which has a long isolation cycle and high cost, and is not suitable for large-scale testing at the grassroots level. The electron microscopy method requires more advanced equipment and higher cost investment, and is not suitable for large-scale sample testing and wide promotion. The disadvantages of antigen detection technology are poor repeatability; it is easily interfered by autoantibodies, heterophilic antibodies, etc., and false positives are prone to occur. PCR detection technology requires endpoint detection and requires opening the cover for gel electrophoresis, which is very easy to form aerosol contamination, affecting subsequent detection, and the operation is cumbersome. The total detection time is close to 3 hours; and there is non-specific amplification. When the non-specific band is the same size as the target band, it cannot be distinguished. The disadvantages of loop-mediated isothermal amplification technology are: the requirements for primers are particularly high, and the amplified product cannot be used for cloning and sequencing; due to its high sensitivity, it forms aerosols, causing false positives, affecting the test results, and because the amplified product fragments are large, it is difficult to remove aerosol contamination after forming.

[0005] Therefore, further research on the detection methods of bovine rotavirus is still necessary. Summary of the invention

[0006] One of the purposes of the present invention is to provide a bovine rotavirus nucleic acid detection method and application with good specificity and sensitivity.

[0007] In order to achieve this object, the technical solution of the present invention is as follows:

[0008] The present invention provides a bovine rotavirus RAA-CRISPR / CAS reaction system, which comprises: bovine rotavirus RAA upstream and downstream primers and crRNA; the sequences of the bovine rotavirus RAA upstream and downstream primers are respectively shown in SEQ ID NO.1-2, and the sequence of the crRNA is shown in SEQ ID NO.3.

[0009] Recombinase-mediated strand replacement nucleic acid amplification (RAA) is a constant temperature rapid nucleic acid amplification technology that uses recombinases obtained from bacteria or fungi. At room temperature, the recombinase can bind tightly to the primer DNA to form a polymer of enzyme and primer. When the primer searches for a complementary sequence that completely matches it on the template DNA, it opens the double-stranded structure of the template DNA with the help of single-stranded binding proteins, and forms a new complementary DNA chain under the action of DNA polymerase, and the amplified product grows exponentially.

[0010] The CRISPR / Cas system is derived from the adaptive immune system of bacteria and archaea. Under the guidance of CRISPR RNA (crRNA) or guide RNA (single-guide RNA, sgRNA), some Cas proteins have trans-splicing activity that can be activated by the target in addition to the cis-splicing activity that recognizes and splices specific targets. That is, after these Cas proteins and crRNA specifically recognize the target, they can be activated to perform trans-splicing activity against ssDNA or ssRNA. Therefore, the CRISPR / Cas system has the potential to detect nucleic acids.

[0011] The present invention obtains a bovine rotavirus nucleic acid detection method based on RAA and CRISPR / CAS technology research, which has strong specificity and high sensitivity and can effectively and quickly realize the detection of bovine rotavirus.

[0012] The bovine rotavirus RAA-CRISPR / CAS reaction system of the present invention further comprises: ssDNA and LbCas12a protein, the sequence of the ssDNA is shown in SEQ ID NO.10, and the ssDNA is labeled with a fluorescent group;

[0013] The fluorescent group includes a fluorescent reporter group and a fluorescent quencher group; the fluorescent reporter group is any one selected from FAM, HEX, JOE, TET, CY3, CY5, ROX, and Texas; the fluorescent quencher group is any one selected from TAMRA, BHQ1, BHQ2, BHQ3, MGB, and DABCYL.

[0014] Preferably, the concentration ratio of the LbCas12a protein to crRNA is (15-55): (50-250), more preferably 25:100.

[0015] The present invention explores the concentration ratio of LbCas12a protein to crRNA in the reaction system, and finds that the detection effect is better by adopting the above-mentioned limited ratio of the present invention.

[0016] The bovine rotavirus RAA-CRISPR / CAS reaction system of the present invention specifically includes: 2-5 mmol / L dNTP, 0.5-1.5 μmol / L single-stranded binding protein, 1.0-2.5 μmol / L recombinase protein, 0.2-1.0 U / μl DNA polymerase, 15-50 mmol / L TrisHCl, 20-80 mmol / L NaCl, 5-15 mmol / L dithiothreitol, 0.8-5.0 μmol / L bovine rotavirus RAA upstream primer, 0.8-5.0 μmol / L bovine rotavirus RAA downstream primer, 0.5-3 μmol / L crRNA, 0.1-1.0 μmol / L LbCas12a, 0.2-1.5 U / μL Exo exonuclease, 20-80 nmol / L ssDNA, and 0.2-1.0 U / μL Rnase Inhibitor.

[0017] The present invention also provides a set of bovine rotavirus RAA detection primer pairs, and the sequences of the bovine rotavirus RAA detection primer pairs are shown in SEQ ID NO.1-2 respectively.

[0018] The present invention further provides a crRNA molecule, whose sequence is shown in SEQ ID NO.3.

[0019] The present invention further provides the use of the above-mentioned bovine rotavirus RAA-CRISPR / CAS reaction system or the bovine rotavirus RAA detection primer pair or crRNA molecule in detecting bovine rotavirus for non-disease diagnosis or treatment purposes or in preparing lyophilized powder, lyophilized liquid, lyophilized tube or kit for bovine rotavirus detection.

[0020] The bovine rotavirus RAA-CRISPR / CAS reaction system provided by the present invention can be prepared as a detection reagent or a kit. For example, for ease of use, the bovine rotavirus RAA-CRISPR / CAS reaction system provided by the present invention can be prepared into a bovine rotavirus one-tube nucleic acid detection lyophilized liquid, lyophilized powder or lyophilized tube.

[0021] The present invention further provides a method for detecting bovine rotavirus for purposes other than disease diagnosis or treatment, which uses the above-mentioned bovine rotavirus RAA-CRISPR / CAS reaction system to amplify the nucleic acid of a sample to be tested, and if an amplification curve appears or the amplification product appears as white light under an ultraviolet lamp, it is determined that the sample to be tested contains bovine rotavirus; or, the above-mentioned bovine rotavirus RAA detection primer pair is used to amplify the nucleic acid of the sample to be tested, and the amplification product is detected by agarose gel electrophoresis. If a band appears, it is determined that the sample to be tested contains bovine rotavirus.

[0022] In the present invention, when the bovine rotavirus RAA-CRISPR / CAS reaction system is used to amplify the nucleic acid of the sample to be tested, the procedure is preferably: 38° C., 60-100 s preheating for 1 cycle; 38-40° C., 30 s amplification, 40 cycles.

[0023] Preferably, when performing the detection amplification reaction, 50 μL of the lyophilized liquid containing the bovine rotavirus RAA-CRISPR / CAS reaction system is freeze-dried, and then 44.5 μL of the premixed solution Buffer A (the ingredients are as follows: 10-50% polyethylene glycol); 2.5 μL of the premixed solution Buffer B (the ingredients are as follows: 150-500 mmol / L magnesium acetate and 10-150 mmol / L magnesium chloride) are added for remelting, the template is 3 μL, and the total reaction system is 50 μL.

[0024] When the bovine rotavirus RAA detection primer pair is used for nucleic acid detection of the sample to be tested, the reaction system includes: 2-5 mmol / L dNTP, 0.5-1.5 μmol / L single-stranded binding protein, 1.0-2.5 μmol / L recombinase protein, 0.2-1.0 U / μl DNA polymerase, 15-50 mmol / L TrisHCl, 20-80 mmol / L NaCl, 5-15 mmol / L dithiothreitol, 0.8-5.0 μmol / L bovine rotavirus RAA upstream primer, 0.8-5.0 μmol / L bovine rotavirus RAA downstream primer, 150-500 mmol / L magnesium acetate and 10-150 mmol / L magnesium chloride. The reaction temperature is 35-40°C and the reaction time is 30-60 minutes.

[0025] The detection method of the present invention has various interpretation modes, which can be flexibly selected according to the actual detection scenario, which is conducive to promotion and application.

[0026] The present invention also provides a bovine rotavirus RAA detection lyophilized liquid, which comprises: the bovine rotavirus RAA-CRISPR / CAS reaction system, trehalose with a final concentration of 10-20 mmol / L, mannitol with a final concentration of 20-50 mmol / L, and bovine serum albumin with a final concentration of 5-15%.

[0027] The present invention further provides a bovine rotavirus RAA detection freeze-dried powder, which comprises components obtained by freeze-drying the above-mentioned bovine rotavirus RAA detection freeze-dried liquid; the freeze-drying step comprises: freezing the bovine rotavirus RAA detection freeze-dried liquid at -80°C for 1-2 hours and then entering a pre-freezing stage, the pre-freezing stage comprising: a pre-freezing stage, equipment vacuuming, freeze drying and analytical drying stages; the conditions of the analytical drying stage are: raising the partition temperature to -20--25°C and maintaining it for 12-16 hours, then raising the partition temperature to 4-8°C and maintaining it for 0.5-1 hour, and finally lowering the partition temperature to 20-25°C and maintaining it for 3-6 hours.

[0028] The bovine rotavirus RAA detection freeze-dried powder of the invention is obtained by freeze-drying a bovine rotavirus RAA detection freeze-dried liquid, and the freeze-dried tube is obtained by freeze-drying the freeze-dried liquid and fixing it in a tubular container.

[0029] When preparing the freeze-dried tube of the present invention, it is preferred that 50 μL of bovine rotavirus RAA detection freeze-dried solution (the volume is supplemented with nuclease-free water) is placed in the container and then the freeze-drying operation is performed.

[0030] The present invention further provides a bovine rotavirus RAA detection kit, which includes the above-mentioned bovine rotavirus RAA-CRISPR / CAS reaction system or bovine rotavirus RAA detection primer pair or crRNA molecule or bovine rotavirus RAA detection lyophilized liquid or bovine rotavirus RAA detection lyophilized powder.

[0031] The beneficial effects of the present invention are at least:

[0032] (1) The present invention provides isothermal amplification. The RAA-CRISPR-Cas12a system can be performed at room temperature, with an optimum temperature of 37 to 42°C. It does not require expensive temperature cycling equipment, thus reducing costs.

[0033] (2) The detection method provided by the present invention is time-saving. The entire reaction process can be completed within 10 to 20 minutes, and no denaturation is required, thereby achieving rapid detection of bovine rotavirus.

[0034] (3) The detection method provided by the present invention has high sensitivity, and the minimum detection limit reaches 250 copies / mL. The RAA-CRISPR-Cas12a system maintains high sensitivity in a short reaction time, and can obtain about 10 copies / mL from a single template molecule.12 Order of magnitude amplification product.

[0035] (4) The detection method provided by the present invention has high specificity. The RAA-CRISPR-Cas12a system only recognizes and cuts the target sequence and does not react to non-target sequences.

[0036] (5) The one-tube nucleic acid detection reagent prepared by the present invention can be stored at room temperature. 2+ In addition, the remaining system components are stored in the reaction tube in a dry powder state, which is stable and easy to store and transport, easy to operate, and reduces the possibility of contamination in the closed reaction system.

[0037] (6) The detection method provided by the present invention has diversified reading results. The results of the RAA-CRISPR-Cas12a system can be detected by agarose gel electrophoresis, the amplification process can be monitored in real time by real-time PCR, and can also be visualized under ultraviolet light.

[0038] The present invention uses the RAA-CRISPR-Cas12a reaction system to construct a fluorescence detection and power-free on-site visual detection method for bovine rotavirus genes. The results can be determined by fluorescence detection or by naked eyes within 20 minutes. The detection equipment is small, the cost is low, the whole process is sealed, and no aerosol pollution is generated. Its application can help control the prevalence of bovine rotavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the detection result of bovine rotavirus nucleic acid by bovine rotavirus RAA-CRISPR-Cas12a in Example 2 of the present invention. In the figure, 1 is bovine rotavirus fecal nucleic acid, and 2 is a negative control (nuclease-free water).

[0040] Figure 2 This is a photograph of the RPA-CRISPR-CAS12a product excited under 302 nM ultraviolet light in Example 2 of the present invention. In the figure, 1 is bovine rotavirus fecal nucleic acid, and 2 is a negative control (nuclease-free water).

[0041] Figure 3 This is the specific detection result of bovine rotavirus RAA-CRISPR-Cas12a detecting bovine rotavirus and other pathogens in Example 3 of the present invention. In the figure, 1 is bovine rotavirus; 2 is coronavirus; 3 is coccidia; 4 is Cryptosporidium; 5 is Clostridium perfringens F type; 6 is Escherichia coli; 7 is Shigella dysenteriae; and 8 is Salmonella.

[0042] Figure 4This is the specific test result of bovine rotavirus and other pathogens under ultraviolet light by RPA-CRISPR-CAS12a in Example 3 of the present invention. In the figure, 1 is bovine rotavirus; 2 is coronavirus; 3 is coccidia; 4 is Cryptosporidium; 5 is Clostridium perfringens F type; 6 is Escherichia coli; 7 is Shigella dysenteriae; and 8 is Salmonella.

[0043] Figure 5 This is the specific detection result of bovine rotavirus RAA-CRISPR-Cas12a in Example 3 of the present invention for detecting the VP2 gene of bovine rotavirus and rotavirus of other species. In the figure, 1 is bovine rotavirus; 2 is mouse rotavirus; 3 is human rotavirus; 4 is porcine rotavirus; 5 is avian rotavirus; 6 is rabbit rotavirus; 7 is feline rotavirus; and 8 is equine rotavirus.

[0044] Figure 6 This is the specific test result of the VP2 gene of bovine rotavirus and other species rotavirus under ultraviolet light by bovine rotavirus RPA-CRISPR-CAS12a in Example 3 of the present invention. In the figure, 1 is bovine rotavirus; 2 is mouse rotavirus; 3 is human rotavirus; 4 is porcine rotavirus; 5 is avian rotavirus; 6 is rabbit rotavirus; 7 is feline rotavirus; and 8 is equine rotavirus.

[0045] Figure 7 is the sensitivity test result of bovine rotavirus RAA-CRISPR-Cas12a in Example 4 of the present invention. In the figure, 1 is 1×10 8 copies / mL; 2 is 1×10 7 copies / mL; 3 is 1×10 6 copies / mL; 4 is 1×10 5 copies / mL; 5 is 1×10 4 copies / mL; 6 is 1×10 3 copies / mL; 7 is 500 copies / mL; 8 is 250 copies / mL; 9 is 125 copies / mL; 10 is nuclease-free water.

[0046] Figure 8 is the sensitivity test result of bovine rotavirus RPA-CRISPR-CAS12a under ultraviolet light in Example 4 of the present invention. In the figure, 1 is 1×10 8 copies / mL; 2 is 1×10 7 copies / mL; 3 is 1×10 6 copies / mL; 4 is 1×10 5 copies / mL; 5 is 1×10 4 copies / mL; 6 is 1×103 copies / mL; 7 is 500 copies / mL; 8 is 250 copies / mL; 9 is 125 copies / mL; 10 is nuclease-free water.

[0047] Fig. 9 This is the intra-batch reproducibility result of bovine rotavirus RAA-CRISPR / CAS in Example 5 of the present invention. In the figure, 1-3 is the concentration of 1×10 7 copies / mL of bovine rotavirus plasmid, and 4 is nuclease-free water.

[0048] Fig.10 The bovine rotavirus RAA-CRISPR / CAS in Example 5 of the present invention is 1×10 7 The results of intra-batch reproducibility test of positive plasmids with a concentration of 1×10 7 copies / mL of positive plasmid, and 4 is nuclease-free water.

[0049] Fig.11 The results of the batch-to-batch reproducibility of bovine rotavirus RAA-CRISPR / CAS in Example 5 of the present invention are shown in Figure 1-3. The concentration of the three batches tested was 1×10 7 copies / mL of bovine rotavirus plasmid was used as template and 4 was nuclease-free water.

[0050] Fig.12 The bovine rotavirus RAA-CRISPR / CAS in Example 5 of the present invention is 1×10 7 The results of the inter-batch reproducibility test of three batches of positive plasmids with a concentration of 1×10 7 copies / mL of positive plasmid, and 4 is nuclease-free water.

[0051] Fig.13 This is the screening result of bovine rotavirus RAA primers in comparative experiment 1 of the present invention. In the figure, 1 is the positive plasmid detected by the BRV-F and BRV-R primer pairs; 2 is the nuclease-free water detected by the BRV-F and BRV-R primer pairs; 3 is the positive plasmid detected by the BRV-F1 and BRV-R1 primer pairs; 4 is the nuclease-free water detected by the BRV-F1 and BRV-R1 primer pairs; 5 is the positive plasmid detected by the BRV-F2 and BRV-R2 primer pairs; 6 is the nuclease-free water detected by the BRV-F2 and BRV-R2 primer pairs.

[0052] Fig.14This is the result of crRNA screening of bovine rotavirus in comparative experiment 2 of the present invention. In the figure, 1 is a positive plasmid for BRV-crR detection; 2 is a positive plasmid for BRV-crR1 detection; 3 is a positive plasmid for BRV-crR2 detection; 4 is nuclease-free water for BRV-crR detection; 5 is nuclease-free water for BRV-crR1 detection; 6 is nuclease-free water for BRV-crR2 detection.

[0053] Fig.15 This is the test result of bovine rotavirus crRNA screening under ultraviolet light in comparative experiment 2 of the present invention. The information represented by 1-6 in the figure is Fig.14 Same as in. DETAILED DESCRIPTION

[0054] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field, or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels or prepared according to conventional methods in the field.

[0056] The freeze-dried liquid components of the bovine rotavirus RAA-CRISPR / CAS reaction system in the present invention are as follows: 2-5 mmol / L dNTP, 0.5-1.5 μmol / L single-stranded binding protein, 1.0-2.5 μmol / L recombinase protein, 0.2-1.0 U / μl DNA polymerase, 15-50 mmol / L TrisHCl, 20-80 mmol / L NaCl, 5-15 mmol / L dithiothreitol, 0.8-5.0 μmol / L bovine rotavirus RAA upstream primer, 0.8-5.0 μmol / L bovine rotavirus RAA downstream primer, 0.5-3 μmol / L crRNA, 0.1-1.0 μmol / L LbCas12a, 0.2-1.5 U / μl Exo exonuclease, 20-80 nmol / L ssDNA, 0.2-1.0 U / μl RNase Inhibitor, 10-20mmol / L trehalose, 20-50mmol / L mannitol, 5-15% bovine serum albumin.

[0057] After the lyophilized liquid is freeze-dried, it is reconstituted with 44.5 μL of premixed buffer A (the ingredients are as follows: 10-50% polyethylene glycol); 2.5 μL of premixed buffer B (the ingredients are as follows: 150-500 mmol / L magnesium acetate and 10-150 mmol / L magnesium chloride), 3 μL of template, and the total reaction system is 50 μL.

[0058] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0059] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0060] Example 1 Bovine rotavirus RAA-CRISPR / CAS primer design

[0061] According to the VP2 gene JX971570.1 of bovine rotavirus registered in Genbank, RAA primers, crRNA primers, and ssDNA primers were designed. The designed primer sequences are shown in Table 1 and were synthesized by a gene synthesis company.

[0062] Table 1

[0063]

[0064] Example 2 Establishment of a bovine rotavirus RAA-CRISPR / CAS detection method

[0065] This embodiment provides a bovine rotavirus RAA-CRISPR / CAS detection method, the method comprising:

[0066] (1) Preparation of freeze-dried powder for nucleic acid detection in one tube:

[0067] One tube of bovine rotavirus RAA-CRISPR CAS12a freeze-dried reaction solution (nucleic acid detection freeze-dried solution), a total of 50μL, including: 3mmol / L dNTP, 1.0μmol / L single-stranded binding protein, 1.3μmol / L recombinase protein, 0.5U / μL DNA polymerase, 25mmol / L TrisHCl, 40mmol / L NaCl, 10mmol / L dithiothreitol, 4μmol / L bovine rotavirus RAA upstream primer, 4μmol / L bovine rotavirus RAA downstream primer, 1.0μmol / L crRNA, 0.25μmol / LLbCas12a, 1U / μL Exo exonuclease, 50nmol / L ssDNA, 0.5U / μL RNase Inhibitor, 15mmol / L trehalose, 25mmol / L mannitol, 8% bovine serum albumin and nuclease-free water (to be supplemented to 50μL) to obtain a bovine rotavirus one-tube nucleic acid detection lyophilized solution. The lyophilized solution was placed in 0.2mL eight-row PCR tubes for lyophilization.

[0068] The lyophilized liquid was first frozen at -80°C for 1 hour and then entered the pre-freezing stage. The freeze-drying condition of the equipment was that the partition temperature dropped to -55°C and the pre-freezing holding time was 40 minutes. Then the equipment was evacuated and freeze-dried for 1 hour. After the pre-freezing stage, analytical drying was carried out, the partition temperature was raised to -25°C and maintained for 15 hours, and then the partition temperature was raised to 5°C and maintained for 0.5 hours, and finally the partition was lowered to 25°C and maintained for 5 hours. A tube of bovine rotavirus RAA-CRISPR CAS12a lyophilized tube containing bovine rotavirus RAA-CRISPR CAS12a lyophilized powder was obtained.

[0069] (2) Genome extraction:

[0070] The sample to be tested is subjected to nucleic acid extraction using a commercial nucleic acid extraction kit to obtain the nucleic acid to be tested; in this embodiment, a fecal swab infected with bovine rotavirus (whose VP2 gene has a homology rate of 100% with the RVA / Bovine-tc / KOR / KJ44 / 2006 / G5P[1] strain (Genbank: MF940702.1)) is used as the sample to be tested.

[0071] (3) RAA-CRISPR-Cas12a amplification

[0072] To a bovine rotavirus RAA-CRISPR CAS12a freeze-dried tube, add 44.5 μL of premixed solution Buffer A (30% polyethylene glycol) to reconstitute, then add 3 μL of the nucleic acid to be tested, and then add 2.5 μL of Buffer B (250 mmol / L magnesium acetate and 120 mmol / L magnesium chloride) solution on the tube cap. After covering the tube cap of the reaction tube, simply rotate and centrifuge Buffer B to the bottom of the freeze-dried tube, a total of 50 μL system, mix evenly, and perform amplification reaction. The amplification program is: 38°C, 60s preheating; 38°C, 30s amplification, 40 cycles, to obtain nucleic acid amplification products.

[0073] RAA-CRISPR-Cas12a amplification results:

[0074] Results of the one-tube bovine rotavirus RAA-CRISPR CAS12a nucleic acid test are available at Figure 1 , bovine rotavirus nucleic acid has an amplification curve, while the negative control has no amplification curve, indicating that the method provided by the present invention can detect whether bovine rotavirus exists in a sample.

[0075] The RAA-CRISPR-Cas12a amplification reaction was excited by a gel imager at 302nM UV light, and the results were read by naked eyes and photographed. Figure 2 As shown, the reaction tube containing bovine rotavirus nucleic acid product appears white light, while the negative control reaction tube appears colorless.

[0076] Example 3 Specificity test of bovine rotavirus RAA-CRISPR / CAS detection method

[0077] This example verifies the specificity of the provided bovine rotavirus RAA-CRISPR / CAS detection method.

[0078] In this example, DNA / RNA of bovine rotavirus, coronavirus, coccidia, cryptosporidium, Clostridium perfringens F type, Escherichia coli, Shigella dysenteriae, and Salmonella strains recorded in Example 2 were respectively extracted as nucleic acids to be tested, and RAA-CRISPR-Cas12a amplification detection was performed. The one-tube bovine rotavirus RAA-CRISPR CAS12a lyophilized tube, premix Buffer A and Buffer B used were as described in Example 2.

[0079] RAA-CRISPR-Cas12a amplification detection was performed on rotavirus VP2 gene synthetic plasmids of seven common species, including mice, humans, pigs, poultry, rabbits, cats, and horses, and bovine rotavirus nucleic acid. The one-tube bovine rotavirus RAA-CRISPR CAS12a lyophilized tube, premix Buffer A and Buffer B used were as described in Example 2.

[0080] The reaction system is as follows: add 44.5 μL Buffer A and 3 μL nucleic acid to be tested to a bovine rotavirus RAA-CRISPR CAS12a freeze-dried tube, then add 2.5 μL Buffer B solution to the tube cap, cover the freeze-dried tube cap, and simply rotate to centrifuge Buffer B to the bottom of the freeze-dried tube, a total of 50 μL system, mix evenly, and use it as a reaction tube for amplification reaction. Amplification program: 38°C, 100s preheating; 40°C, 30s amplification, 40 cycles; in the reaction tube, a nucleic acid amplification product is obtained.

[0081] The amplification results of each reaction tube are shown in Figure 3 , Figure 5 ,Depend on Figure 3 The results show that the fluorescence intensity signal is obtained only in the reaction tube of bovine rotavirus, while the reaction tubes of other types of pathogens (coronavirus, coccidia, cryptosporidium, Clostridium perfringens F type, Escherichia coli, Shigella dysenteriae, and Salmonella strains) have no amplification curves. This shows that the RPA-CRISPR-CAS12a detection method provided in this example has better detection specificity for bovine rotavirus.

[0082] Bovine rotavirus RPA-CRISPR-CAS12a UV-specific test results are shown in Figure 4 , Figure 6 ,Depend on Figure 4 It can be seen that the detection method of RAA-CRISPR / Cas12a can only detect bovine rotavirus as white light, but no amplification is found for other types of pathogens (coronavirus, coccidia, cryptosporidium, Clostridium perfringens F type bacteria, Escherichia coli, Shigella dysenteriae, and Salmonella strains), and it appears colorless and transparent under ultraviolet light, indicating that nonspecific amplification cannot occur. The detection method has good specificity for bovine rotavirus detection.

[0083] Depend on Figure 5 The results show that the fluorescence intensity signal was obtained only in the reaction tube of bovine rotavirus, while the reaction tubes of rotavirus VP2 gene of other species (mouse, human, pig, poultry, rabbit, cat, horse) had no amplification curve. This shows that the RPA-CRISPR-CAS12a detection method provided in this example has better detection specificity for bovine rotavirus.

[0084] Depend on Figure 6 It can be seen that the RAA-CRISPR / Cas12a detection method can only detect bovine rotavirus as white light, while the rotavirus VP2 gene of other species (mouse, human, pig, poultry, rabbit, cat, horse) has no amplification and appears colorless and transparent under ultraviolet light, indicating that nonspecific amplification cannot occur. The detection method has good specificity for the detection of the VP2 gene of bovine rotavirus.

[0085] Example 4 Sensitivity test of bovine rotavirus RAA-CRISPR / CAS detection method

[0086] In this example, the concentration is 1×10 8 The bovine rotavirus plasmid (introduced with the VP2 gene JX971570.1) with a concentration of 10 copies / mL was diluted 10-fold. 3 The plasmid with 1×10 copies / mL was diluted 2-fold. 8 copies / mL~10 3 The sensitivity of the RAA-CRISPR-Cas12a reagent was tested using plasmids of bovine rotavirus at gradient concentrations of 500 copies / mL, 250 copies / mL, and 125 copies / mL as templates and nuclease-free water as a negative control. The reaction conditions and reaction system were the same as in Example 2.

[0087] Bovine rotavirus RAA-CRISPR-Cas12a sensitivity test results are shown in Figure 7 , it can be seen that the minimum detection limit of the RAA-CRISPR / Cas12a detection method for bovine rotavirus is 250 copies / mL, and the detection method has the characteristic of high sensitivity.

[0088] Bovine rotavirus RPA-CRISPR-CAS12a sensitivity test results under UV light are shown in Figure 8 , it can be seen that the RAA-CRISPR / Cas12a detection method can detect white light for bovine rotavirus samples with a concentration of more than 250 copies / mL, but cannot detect white light for samples with a concentration of 125 copies / mL, indicating that the minimum detection limit of the present invention is 250 copies / mL. The detection method has the characteristic of high sensitivity.

[0089] Example 5 Repeatability test of bovine rotavirus RAA-CRISPR / CAS detection method

[0090] This example provides a repeatability analysis of a bovine rotavirus RAA-CRISPR / CAS detection method, the steps are as follows:

[0091] Select a known positive sample and perform intra-batch repeat detection and inter-batch repeat detection respectively. Intra-batch repeat detection: perform 3 repeats of a known positive sample in the same batch of experiments. Inter-batch repeat experiment: perform 3 batches of a known positive sample. The reaction conditions and reaction system are the same as in Example 2.

[0092] The intra-batch reproducibility results of bovine rotavirus RAA-CRISPR / CAS are shown in Fig. 9, it can be seen that the RAA-CRISPR / Cas12a detection method is effective for 1×10 7 The bovine rotavirus with 100 copies / mL has an amplification curve, and the fluorescence signal and CT value within the batch are basically consistent. The detection method has good intra-batch repeatability.

[0093] Bovine rotavirus RAA-CRISPR / CAS under UV light 1×10 7 The results of intra-batch reproducibility test of positive plasmids with 10 copies / mL are shown in Fig.10 . It can be seen that bovine rotavirus RAA-CRISPR / CAS can effectively inhibit the growth of 1×10 7 White light can be detected for all positive plasmids with 10 copies / mL, indicating that the detection method has good intra-batch repeatability.

[0094] The results of the batch-to-batch reproducibility of bovine rotavirus RAA-CRISPR / CAS are shown in Fig.11 , it can be seen that the three batches of RAA-CRISPR / Cas12a detection reagents are 1×10 7 The fluorescent intensity signals of bovine rotavirus with 100 copies / mL were obtained, and the fluorescent signals and CT values ​​between batches were basically consistent. The detection method has the characteristics of strong batch repeatability.

[0095] Bovine rotavirus RAA-CRISPR / CAS under UV light 1×10 7 The results of the inter-batch reproducibility test of positive plasmids with a total number of copies / mL are shown in Fig.12 . It can be seen that the three batches of bovine rotavirus RAA-CRISPR / CAS positive plasmid 1×10 7 White light can be detected for all copies / mL, indicating that the detection method has good inter-batch repeatability.

[0096] The above results indicate that the bovine rotavirus RAA-CRISPR / CAS detection method provided by the present invention has good repeatability within and between batches.

[0097] Comparative experiment 1 Screening of the best RAA primers

[0098] Using the bovine rotavirus plasmid described in Example 4 as a template, the three pairs of designed RAA primers (Table 2) were added to the amplification system respectively. The specific reaction system is shown in Table 3 below. 2.5 μL of magnesium acetate with a final concentration of 250 mmol / L and magnesium chloride reagent with a final concentration of 120 mmol / L were added to the bottle cap, all the reaction reagents were mixed and centrifuged, and immediately placed in a 37°C metal bath after centrifugation and incubated for 30 minutes. After the incubation, a 2% gel was used for electrophoresis experiment. The gel results were excited by a UV lamp at 302 nM, and the results were judged by naked eyes and photographed. The results are shown in Fig.13The results showed that RAA primers BRV-F and BRV-R had target bands at around 216bp and had the best specificity. RAA primers BRV-F1 and BRV-R1 and RAA primers BRV-F2 and BRV-R2 all had non-specific bands.

[0099] Table 2 RAA primer list

[0100]

[0101]

[0102] Table 3 RAA system formulation table

[0103] system Final concentration dNTP 3mmol / L Single-chain binding protein 1.0μmol / L Recombinase protein 1.3μmol / L DNA polymerase 0.5U / μL TrisHCl 25mmol / L NaCl 40mmol / L Dithiothreitol 10mmol / L RAA upstream and downstream primers 4μmol / L RAA upstream and downstream primers 4μmol / L template 3μL Nuclease-free water Make up to 50 μL

[0104] Comparative experiment 2 optimal crRNA screening

[0105] The bovine rotavirus plasmid described in Example 4 was used as a template, nuclease-free water was used as a negative control, RAA upstream and downstream primers (SEQ ID NO. 1 and 2) were combined with ssDNA probes, and BRV-crR, BRV-crR1 and BRV-crR2 (Table 4) were used for RAA-CRISPR / CAS fluorescence detection. The specific reaction system is shown in Table 5 below. 2.5 μL of a final concentration of 250 mmol / L magnesium acetate and 120 mmol / L magnesium chloride reagent were added to the bottle cap, and all the reaction reagents were mixed to form the CRISPR / Cas12a initial detection system for centrifugation. After centrifugation, fluorescence amplification detection was performed immediately, and the FAM channel was selected. The amplification program: 38°C, 100s preheating; 40°C, 30s amplification, 40 cycles; after the end, the data was saved for fluorescence signal analysis, and the results were as follows Fig.14 shown.

[0106] The RAA-CRISPR-Cas12a amplification reaction was excited by a gel imager at 302nM UV light, and the results were interpreted by naked eyes and photographed. Fig.15 By comparing the Ct value and fluorescence intensity, the best crRNA was selected as BRV-crR.

[0107] Table 4 crRNA sequence list

[0108]

[0109] Table 5 RAA-CRISPR CAS12a system preparation table

[0110]

[0111]

[0112] Comparative experiment 3 Selection of optimal concentrations of crRNA and CAS12a protein

[0113] The bovine rotavirus plasmid recorded in Example 4 was used as a template, nuclease-free water was used as a negative control, and the optimal RAA upstream and downstream primers and the optimal crRNA were combined with ssDNA probes for RAA-CRISPR / CAS fluorescence detection. The specific reaction system is shown in Table 6 below, where the final concentration of crRNA is X and the final concentration of Cas12a is Y, X and Y are variables, and the volume of LbCas12a protein and crRNA required to be added to different concentrations needs to be calculated with a total reaction volume of 50 μL, and the final concentrations of other components remain unchanged.

[0114] The selected LbCas12a protein concentrations were 0.15μM, 0.25μM, 0.35μM, 0.45μM, and 0.55μM, respectively; the selected crRNA concentrations were 0.5μM, 1.0μM, 1.5μM, 2.0μM, and 2.5μM for cross-reaction. 2.5μL of magnesium acetate with a final concentration of 250mmol / L and magnesium chloride reagent with a final concentration of 120mmol / L were added to the bottle cap, all the reaction reagents were mixed evenly, centrifuged, and the amplification reaction was performed immediately. The reporter molecule selected was a fluorescent reporter molecule, and the amplification program was 38°C, 60s preheating for 1 cycle; 38°C, 30s amplification, 40 cycles. The nucleic acid amplification product was obtained, and the Ct value data after the reaction was completed was saved and analyzed. The results are shown in Table 7. The experimental results were analyzed and the final concentration of LbCas12a protein in the optimal reaction system was determined to be 0.25μM, and the final concentration of crRNA was 1.0μM.

[0115] Table 6 RAA-CRISPR CAS12a system preparation table

[0116]

[0117]

[0118] Table 7 Screening results of the ratio of LbCas12a to crRNA

[0119]

[0120] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A bovine rotavirus RAA-CRISPR / CAS reaction system, characterized in that: include: Bovine rotavirus RAA upstream and downstream primers and crRNA; the sequences of the bovine rotavirus RAA upstream and downstream primers are shown in SEQ ID NO.1-2, respectively, and the sequence of the crRNA is shown in SEQ ID NO.3; The bovine rotavirus RAA-CRISPR / CAS reaction system further comprises: ssDNA and LbCas12a protein, the sequence of the ssDNA is shown in SEQ ID NO.10, and the ssDNA is labeled with a fluorescent group; The concentration ratio of the LbCas12a protein to crRNA is (15~55): (50~250).

2. The bovine rotavirus RAA-CRISPR / CAS reaction system according to claim 1, characterized in that: The concentration ratio of the LbCas12a protein to crRNA is 25:

100.

3. The bovine rotavirus RAA-CRISPR / CAS reaction system according to any one of claims 1 to 2, characterized in that: include: 2~5 mmol / L dNTP, 0.5~1.5 μmol / L single-stranded binding protein, 1.0~2.5 μmol / L recombinase protein, 0.2~1.0 U / μl DNA polymerase, 15~50 mmol / L TrisHCl, 20~80 mmol / L NaCl, 5~15 mmol / L dithiothreitol, 0.8~5.0 μmol / L bovine rotavirus RAA upstream primer, 0.8~5.0 μmol / L bovine rotavirus RAA downstream primer, 0.5~3 μmol / L crRNA, 0.1~1.0 μmol / L LbCas12a, 0.2~1.5 U / μL Exo exonuclease, 20~80nmol / L ssDNA, 0.2~1.0 U / μL RNase Inhibitor.

4. Use of the bovine rotavirus RAA-CRISPR / CAS reaction system according to any one of claims 1 to 3 in detecting bovine rotavirus for non-disease diagnosis or treatment purposes or in preparing lyophilized powder, lyophilized liquid, lyophilized tube or kit for bovine rotavirus detection.

5. A method for detecting bovine rotavirus for purposes other than disease diagnosis or treatment, characterized in that: The bovine rotavirus RAA-CRISPR / CAS reaction system described in any one of claims 1 to 3 is used to amplify the nucleic acid of the sample to be tested. If an amplification curve appears or the amplification product appears as white light under ultraviolet light, it is determined that the sample to be tested contains bovine rotavirus.

6. A lyophilized liquid for detecting bovine rotavirus RAA, characterized in that: include: The bovine rotavirus RAA-CRISPR / CAS reaction system according to any one of claims 1 to 3, trehalose with a final concentration of 10-20 mmol / L, mannitol with a final concentration of 20-50 mmol / L, and bovine serum albumin with a final concentration of 5-15%.

7. A lyophilized powder for detecting bovine rotavirus RAA, characterized in that: The invention comprises components obtained by freeze-drying the bovine rotavirus RAA detection freeze-dried liquid as described in claim 6; the freeze-drying step comprises: freezing the bovine rotavirus RAA detection freeze-dried liquid at -80°C for 1-2 hours and then entering a pre-freezing stage, wherein the pre-freezing stage comprises: a pre-freezing stage, equipment vacuuming, freeze drying and analytical drying stages; the conditions of the analytical drying stage are: raising the partition temperature to -20--25°C and maintaining it for 12-16 hours, then raising the partition temperature to 4-8°C and maintaining it for 0.5-1 hour, and finally lowering the partition temperature to 20-25°C and maintaining it for 3-6 hours.

8. A bovine rotavirus RAA detection kit, characterized in that: It comprises the bovine rotavirus RAA-CRISPR / CAS reaction system according to any one of claims 1 to 3, the bovine rotavirus RAA detection lyophilized liquid according to claim 6, or the bovine rotavirus RAA detection lyophilized powder according to claim 7.

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