Rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV)

Abstract

The invention provides a rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV), which comprises the following steps: controlling reaction conditions: the detection method is carried out at 39 DEG C, and the reaction time is 35 minutes; after the amplification reaction of the positive sample is finished, the positive sample is used for showing color change through the HNB color development reaction, and a detection result is judged according to the appearing color change; aiming at the bovine viral diarrhea mucosa disease and the infectious bovine rhinotracheitis virus, two pairs of specific primers are adopted for ensuring the sensitivity and specificity of amplification reaction; after RAA-HNB screening, a positive sample is further verified through nucleic acid electrophoresis. The kit is low in cost, easy and convenient to operate, high in sensitivity, high in specificity and suitable for large-scale screening and field detection, a reliable tool is provided for rapid detection and prevention and control of cattle farm epidemic diseases, and epidemic disease transmission can be controlled in time.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical detection, in particular to a rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV). Background Art

[0002] Bovine viral diarrhea virus (BVDV) and infectious rhinotracheitis virus (IBRV) are two important pathogens that threaten the global beef cattle industry. Infection with these two viruses can cause respiratory diseases, reproductive problems and immunosuppression, causing huge economic losses to the beef cattle industry. Existing detection methods are mostly nucleic acid tests in the laboratory, such as PCR technology. Although it has high sensitivity and specificity, it requires expensive equipment and a long operation process, which is difficult to meet the needs of rapid on-site diagnosis.

[0003] Recombinase-mediated isothermal nucleic acid amplification (RAA) is an emerging molecular detection technology that has the advantages of simple operation, short reaction time, and no need for complex equipment, and is particularly suitable for on-site detection. However, the existing RAA technology needs to be combined with fluorescent probes or other complex detection devices, which is costly and limits its application in large-scale screening. Therefore, the present invention provides a rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV). Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) to solve the problems raised in the above-mentioned background technology. The present invention has the advantages of low cost, simple operation, high sensitivity, strong specificity, etc., and is particularly suitable for large-scale screening and on-site detection.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV), comprising the following steps:

[0006] Step 1: Control reaction conditions: The detection method is carried out at 39°C and the reaction time is 35 minutes;

[0007] Step 2: Perform color development reaction: After the amplification reaction of the positive sample is completed, the HNB color development reaction is used to show the color change, and the test result is judged according to the color change;

[0008] Step 3: For bovine viral diarrhea mucosal disease and bovine infectious rhinotracheitis virus, two pairs of specific primers are used to ensure the sensitivity and specificity of the amplification reaction;

[0009] Step 4: Double verification: After RAA-HNB screening, positive samples were further verified by nucleic acid electrophoresis, where the amplified fragment sizes of infectious bovine rhinotracheitis virus were 385bp and 130bp, and the amplified fragment sizes of infectious bovine rhinotracheitis were 492bp and 359bp.

[0010] Furthermore, under the reaction conditions in step 1, the recombinase, with the assistance of the single-stranded DNA binding protein, binds to the primer to form a complex, quickly recognizes and binds to the target DNA sequence, and forms a D-loop structure.

[0011] Furthermore, it also includes DNA polymerase to carry out chain replication, realize DNA amplification, and make the positive sample show obvious color change quickly from purple to blue, while the negative sample has no color change.

[0012] Furthermore, two pairs of RAA primers were used for each virus to improve the amplification efficiency and specificity of the DNA chain and the sensitivity of the colorimetric detection.

[0013] Furthermore, the specific primers used PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids as templates, and the RAA amplification kit was used to determine the primer amplification time.

[0014] Furthermore, in the process of determining the reaction temperature of RAA-HNB visualization detection, the PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids were used as templates, and the RAA amplification kit was used to determine the primer amplification time.

[0015] Furthermore, in step 4, PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids were used as templates, and gradient dilution was used to determine the range of virus titers for visual detection.

[0016] Furthermore, the reaction conditions of the detection scheme after RAA-HNB visualization optimization included: reaction at 39°C for 35 minutes, observation of the color of the test tube, and verification of the results using nucleic acid electrophoresis.

[0017] Furthermore, it also includes extracting previously positive animal samples during the RAA-HNB visualization positive sample detection process for BVDV and IBRV and testing them using the optimized RAA-HNB scheme.

[0018] Furthermore, the detection conditions of the optimized RAA-HNB protocol were as follows: react at 39°C for 35 minutes, observe the color of the test tube, and verify the results using nucleic acid electrophoresis.

[0019] Beneficial effects of the present invention:

[0020] 1. The rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) has the advantages of simple operation, short detection time, low cost and strong specificity. It is particularly suitable for large-scale on-site screening and can quickly and effectively identify positive samples. The test results can be intuitively judged through the HNB color reaction, and positive samples show obvious color changes. Two pairs of specific primers are used in the detection process of bovine viral diarrhea-mucosal disease and bovine infectious rhinotracheitis to ensure the sensitivity and specificity of the reaction.

[0021] 2. RAA in the rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) does not require complex temperature control equipment, and only needs to react for 35 minutes at 39°C to obtain the results, which greatly shortens the detection time. This mild reaction condition and the characteristics of rapid detection can meet the needs of rapid on-site diagnosis. By using specific primers to detect BVDV and IBRV respectively, the amplification reactions of the two viruses are effectively distinguished, cross-reactions are avoided, and the accuracy of detection is improved. The HNB color reaction further simplifies the interpretation process of the results. Compared with fluorescence or electrophoresis detection methods, the HNB color reaction does not require expensive instruments and equipment, and the color change can be observed by the naked eye (positive reactions show obvious blue), which greatly reduces the technical threshold of the operation. Because the color reaction is combined with RAA amplification, the sensitivity and specificity of the test are significantly improved, ensuring that positive individuals can be accurately identified even in samples with low viral loads.

[0022] 3. This rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) does not require expensive instruments and equipment, and the detection time is greatly shortened. It is suitable for use in beef cattle farms, remote areas or other scenarios with imperfect infrastructure, thus providing an effective tool for the early diagnosis and control of the disease. In addition, the operability and reliability of RAA technology in on-site testing have also been demonstrated. Through simplified operating steps and rapid response time, reliable test results can be obtained in a short time. This convenience is particularly critical for daily monitoring of beef cattle farms and early warning of diseases. It can help farmers take isolation and treatment measures in a timely manner, thereby effectively reducing the spread of diseases and reducing economic losses.

[0023] 4. The RAA technology used in the present invention does not require complex temperature control instruments due to its low-temperature isothermal amplification mechanism, which greatly reduces the usage threshold in terms of time and cost. In addition, the introduction of the HNB color development reaction further shortens the time for reading results, and does not require the use of expensive fluorescent probes or electrophoresis equipment, which makes this method more practical. RAA technology also has certain advantages in temperature requirements and reaction time. The LAMP reaction usually needs to be carried out at a higher temperature of 60-65°C, while RAA can complete the amplification at 39°C, and the reaction speed is faster (results within 35 minutes), which provides more possibilities for simplifying on-site operations and saving time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a schematic structural diagram of the appearance of a rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV) of the present invention;

[0025] Figure 2 Schematic diagram of the principle of RAA-HNB in ​​an embodiment of the present invention;

[0026] Figure 3 The RAA-HNB-BVDV visualization reaction time in the process of determining the RAA-HNB visualization detection reaction time of BVDV and IBRV in the embodiment of the present invention;

[0027] Figure 4 The RAA-HNB-IBRV visualization reaction time in the process of determining the RAA-HNB visualization detection reaction time of BVDV and IBRV in the embodiment of the present invention;

[0028] Figure 5 The RAA-HNB-BVDV visualization reaction time in the process of determining the RAA-HNB visualization detection reaction temperature of BVDV and IBRV in the embodiment of the present invention;

[0029] Figure 6 The RAA-HNB-IBRV visualization reaction time in the process of determining the RAA-HNB visualization detection reaction temperature of BVDV and IBRV in the embodiment of the present invention;

[0030] Figure 7 The range of BVDV copy number detected by RAA-HNB-BVDV visualization in the embodiment of the present invention;

[0031] Figure 8 The range of IBRV copy numbers detected by visual detection of RAA-HNB-IBRV in the embodiment of the present invention;

[0032] Fig. 9 It is a RAA-HNB-IBRV visual sample detection in an embodiment of the present invention;

[0033] Fig.10 It is a RAA-HNB-BVDV visualization sample detection in an embodiment of the present invention;

[0034] Fig.11 These are the BVDV and IBRV PCR products in the examples of the present invention. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0036] See also Figures 1 to 11 The present invention provides the following technical solution: A rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV), comprising the following steps:

[0037] Step 1: Control reaction conditions: The detection method is carried out at 39°C and the reaction time is 35 minutes;

[0038] Step 2: Perform color development reaction: After the amplification reaction of the positive sample is completed, the HNB color development reaction is used to show the color change, and the test result is judged according to the color change;

[0039] Step 3: For bovine viral diarrhea mucosal disease and bovine infectious rhinotracheitis virus, two pairs of specific primers are used to ensure the sensitivity and specificity of the amplification reaction;

[0040] Step 4: Double verification: After RAA-HNB screening, positive samples were further verified by nucleic acid electrophoresis, where the amplified fragment sizes of infectious bovine rhinotracheitis virus were 385bp and 130bp, and the amplified fragment sizes of infectious bovine rhinotracheitis were 492bp and 359bp.

[0041] This example provides a molecular biological identification scheme for BVDV and IBRV viruses, which is as follows:

[0042] 1. Extraction of DNA from nasal swab samples. Take out the sample stored at -80℃, add 200μL PBS to homogenize, then add 20μL QIAGEN protease; add 200μL Buffer-AL, vortex for 15s, and place in a 56℃ water bath for more than 10min; add 200μL ethanol (100%) to the lysed tissue, vortex for 15s; transfer the mixed liquid to the QIAamp Mini column with a pipette, centrifuge at ≥8000rpm and discard the liquid; add 500μL Buffer AW1 to the centrifuge column, centrifuge at 8000rpm for 1min, and discard the liquid; add 500μL Buffer AW2 to the centrifuge column, centrifuge at 14000rpm for 3min, and discard the liquid; take out the centrifuge column and place it in a centrifuge tube in a ventilated place in the clean bench for 10min to completely evaporate the residual alcohol in the column; add 100μL double distilled water, centrifuge and store at -80℃.

[0043] 2. RNA extraction from anal swab samples. Take out the sample stored at -80°C, add 200 μL of lysis buffer (Trizol), and then add 20 μL of QIAGEN protease; add an equal volume of phenol-chloroform-isoamyl alcohol to the lysis mixture and mix thoroughly. Centrifuge at 12,000 rpm for 15 minutes; carefully collect the aqueous phase (upper layer, about 500 μL), add 2.5 times the volume of ice-cold isopropanol to the aqueous phase, and centrifuge at 12,000 rpm for 15 minutes at 4°C; discard the supernatant, retain the RNA precipitate, and gently wash the precipitate with 70% ice-cold ethanol; centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, and dry the precipitate; dissolve the RNA in 30-50 μL of DEPC-treated water or RNase-free water, determine the RNA concentration and purity with NanoDrop, and check the RNA integrity by agarose gel electrophoresis.

[0044] 3. RNA reverse transcription. Use the full-form gold reverse transcription kit. IIProbe One-StepqRT-PCR SuperMix, obtain cDNA template, refer to the instruction manual for details.

[0045] 4. PCR identification of BVDV and IBRV viruses. The extracted DNA / cDNA was used as a template to detect BVDV and IBRV viruses using the PCR detection method. Primer design: PCR detection primers were designed based on the Singer NS3 gene of bovine viral diarrhea virus strain 1 (DQ323915.1), BVDV-up: 5'-GTCGTCCACCTCCAAAAGACAGG-3'; BVDV-Down: 5'-CCTCCTCGTATAGGC TCCAATCG-3'. PCR detection primers were designed based on the glycoprotein G (US4) gene of IBRV (GU591889.1), IBRV-up: 5'-ATGCCTGCCGCCCGGACC-3'; IBRV-Down: 5'-GGTCCGGGCGGCAGGCAT-3'. PCR reaction conditions: 94°C for 5 min, (94°C for 30 s, 58°C for 30 s, 72°C for 2 min) 35 cycles, 72°C for 10 min. Product electrophoresis detection.

[0046] 5. Construction of NS3 / US4 gene positive plasmid. The target gene gel recovery was performed according to the instructions of the Tiangen Company's plasmid extraction kit. Then, the BVDV and IBRV virus PCR products were connected to the cloning vector (Blunt connection system)

[0047]

[0048] Freeze and thaw the DH5α E. coli competent cells on ice, take 5μL of the ligation system and add it to 80μL of DH5α E. coli competent cells, mix well, put on ice for 30min, then keep it in 42℃ water for 90s, quickly take it out and put it on ice for 2min, add 500μL LB culture solution (Blunt vector added to Kan-), and shake and culture at 37℃ for 60min. Take 200μL of culture solution to plate, culture overnight at 37℃, select colonies for PCR detection, expand positive clones, and save the bacterial solution. Then, refer to the instructions of the plasmid extraction kit.

[0049] 6. Establishment of qPCR detection method for BVDV and IBRV viruses. Use Primer Premier 5 to design specific qRT-PCR primers based on BVDV and IBRV viruses. Q-BVDV-up: 5'-AGGAGTGACACTACCAGATTTG-3'; Q-BVDV-Down: 5'-TCCTCGTATAGGCTCCAATCG-3'. Q-IBRV-up: 5'-TCGGCTGGGCTTACCAAG-3'; Q-IBRV-Down: 5'-GCGTCAACCTCAACAAGGG-3'. Detect BVDV and IBRV viruses. The reaction system is as follows:

[0050]

[0051] For the above mixture, the enzyme was activated at 95°C for 5 min; the reaction was carried out at 95°C for 15 s; the reaction was carried out at 56°C for 30 s; the reaction of steps 2-3 was repeated for 35 cycles; the melting curve was measured at 50°C-95°C; and the mixture was cooled at 4°C for 10 s.

[0052] 7. Establishment of RAA detection method for BVDV and IBRV viruses.

[0053] Primer Premier 5 was used to design specific RAA primers for BVDV and IBRV viruses, with 2 pairs of primers for each virus.

[0054] ①-R-BVDV-up:5'-GAACAACTGGCAATTATCGGAA AGATCCAC-3'

[0055] ①-R-BVDV-Down:5'-CAGCCCCGTGTCTACAACCGTGTCCAAATC-3';

[0056] ②-R-BVDV-up:5'-ATTCACGTGTGTCACCGCATCAGGCACACC-3'

[0057] ②-R-BVDV-Down: 5'-CTGCCGCTGCCCTTAATGGTATAAGAACTA-3'.

[0058] ①-R-IBRV-up:5'-TGCGCCCTGGCCCTC TTGGACATGGCGGAGAC-3'

[0059] ①-R-IBRV-Down:5'-CGAGCCCTCGGTCCAGCCCGCAGGGGGTA TTTG-3';

[0060] ②-R-IBRV-up: 5'-ATCGCTTGCTGGTGCTGCTGTT TTCGCTG';

[0061] ②-R-IBRV-Down: 5'-GCTCGTACGCGGGGTTGCTCTTGGCGAACG-3'. RAA Nucleic Acid Amplification Kit (Basic Type) is used for isothermal amplification. The reaction system is as follows:

[0062]

[0063] 8. Establishment of a visualization method for RAA detection of BVDV and IBRV viruses.

[0064] Add 2μL forward primer and 2μL reverse primer to each reaction tube (primer concentration is 10μM, for multiple reactions, step 1 and step 2 can be mixed and then dispensed into the reaction tube); add 12.1μL ddH2O and 2μL nucleic acid template to the reaction tube in sequence (the volume of nucleic acid template added can be adjusted according to the nucleic acid concentration, and the volume of ddH2O added can be adjusted accordingly, until the total volume of template and ddH2O is 14.1μL). Finally, add 2.5μL B buffer and 0.5μL of chromogen hydroxynaphthol blue (HNB) to the reaction tube and mix thoroughly (please be sure to shake the reaction tube upside down 8 to 10 times to mix (you can vortex to mix); for multiple reactions, it is recommended to add B buffer to the inside of the lid of the reaction tube and mix it upside down); after mixing, shake (or quickly centrifuge) the reaction solution to the bottom of the tube, and then immediately put the reaction tube into a constant temperature device for 33-42℃ constant temperature amplification for 25-40 minutes. After the reaction is completed, observe the color. For subsequent nucleic acid gel detection, add 50 μL of phenol / chloroform (1:1) and shake thoroughly. Note that this step can be vigorously shaken and mixed using a vortex shaker. Centrifuge at 12000 rpm for 5 minutes and take 5 μL of supernatant for agarose gel electrophoresis detection.

[0065] 9. Results:

[0066] PCR detection of BVDV and IBRV and construction of virus-positive plasmids. According to the harvested diseased materials, RNA or DNA of the diseased materials were extracted respectively, and the NS3 and US4 genes of BVDV and IBRV were detected by PCR. The results showed that the target band sizes of NS3 and US4 were 1294bp and 1336bp, respectively, which were consistent with expectations. This target fragment was used to construct PCR@-Blunt-BVDV-NS3 plasmid and PCR@-Blunt-BVDV-US4 plasmid, and sequencing confirmed that they were the NS3 and US4 genes of BVDV and IBRV. The results showed that the positive plasmids of the NS3 and US4 genes of BVDV and IBRV were successfully obtained, providing standard positive plasmids for subsequent use.

[0067] This embodiment also provides a process for establishing a RAA-HNB visualization detection method for BVDV and IBRV, which specifically includes the following contents:

[0068] 1. Principle of RAA-HNB

[0069] The recombinase-mediated isothermal nucleic acid amplification technology (RAA) used in this embodiment, combined with the hydroxynaphthol blue (HNB) color reaction, is a rapid, sensitive and suitable nucleic acid amplification method for on-site detection. The basic principle of this technology is that the recombinase, with the assistance of single-stranded DNA binding protein, binds to the primer to form a complex, quickly identifies and binds to the target DNA sequence, and forms a D-loop structure. Subsequently, DNA polymerase performs chain replication to achieve DNA amplification. Under isothermal conditions of 39 degrees Celsius, the reaction time is 35 minutes, and the positive sample will quickly show a significant color change from purple to blue, while the negative sample has no color change. In order to improve the sensitivity of color detection, we used two pairs of RAA primers for each virus, which can improve the amplification efficiency and specificity of the DNA chain, thereby improving the sensitivity of color detection. This method has the advantages of rapidity, sensitivity, specificity and simplicity, can complete the detection in a short time, and can accurately detect the target DNA sequence, making it outstanding in large-scale screening and on-site detection.

[0070] 2. Determination of the reaction time of RAA-HNB visual detection of BVDV and IBRV

[0071] Two pairs of detection primers were designed according to the NS3 and US4 genes of BVDV and IBRV, respectively. Using PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids as templates, the RAA amplification kit was used to determine the primer amplification time. The amplification time at room temperature (37°C) was 25, 30, 35, and 40 minutes, respectively, and then nucleic acid electrophoresis was used for detection. The results showed that the target fragments of the BVDV primers were 492bp and 359bp, respectively, and the best effect was achieved with a room temperature amplification time of 35 minutes. The target fragments of the IBRV primers were 385bp and 130bp, respectively, and the best effect was achieved with a room temperature amplification time of 35 minutes. Figure 3 , Figure 4 3. Determination of reaction temperature for RAA-HNB visual detection of BVDV and IBRV

[0072] Using PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids as templates, the RAA amplification kit was used to determine the primer amplification time. The amplification time at different temperatures (30°C, 33°C, 36°C, 39°C, 42°C) was 35 minutes, followed by nucleic acid electrophoresis for detection. The results showed that the target fragments of the BVDV primers were 492bp and 359bp, respectively, and the reaction temperature of 39°C was optimal. The target fragments of the IBRV primers were 385bp and 130bp, respectively, and the reaction temperature of 39°C was optimal ( Figure 5 , Figure 6 4. The range of viral copy numbers detected by RAA-HNB visualization of BVDV and IBRV

[0073] Using PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids as templates, gradient dilution was performed to make the plasmid template concentrations 2.35x10 1 , 2.35x10 2 , 2.35x10 3 , 2.35x10 4 , 2.35x10 5 , 2.35x10 6 / μL, and explored the range of virus titer for visual detection. The RAA-HNB visualization optimized detection scheme was used, and the reaction was carried out at 39°C for 35 minutes. The color of the test tube was observed, and the results were verified by nucleic acid electrophoresis. The results showed that when the IBRV virus copy number was ≥2.35x10 2 When this scheme is adopted, efficient visual inspection can be performed ( Figure 7 , Figure 8 ).

[0074] 5. RAA-HNB Visualization Positive Sample Detection for BVDV and IBRV

[0075] Take the previously positive animal samples and test them using the optimized RAA-HNB protocol. React at 39°C for 35 minutes, observe the color of the test tube, and use nucleic acid electrophoresis to verify the results. The results show that the RAA-HNB rapid detection protocol established in this project has very good specificity and sensitivity, can quickly screen samples, and can be used in conjunction with laboratory nucleic acid electrophoresis for rapid laboratory verification. It not only greatly reduces the workload and instrument requirements for sample screening, but also reduces time and labor costs, and can be used in conjunction with laboratory nucleic acid electrophoresis verification to solve the work of initial screening and laboratory confirmation at one time.

[0076] This example develops a rapid detection method for bovine viral diarrhea virus (BVDV) and bovine herpes virus (IBRV) based on recombinase-mediated isothermal amplification (RAA) technology, and combines it with hydroxynaphthol blue (HNB) color development reaction to achieve low-cost and simple on-site detection. This method shows significant advantages in sensitivity, specificity and convenience, and is particularly suitable for large-scale screening and resource-constrained environments.

[0077] The application of RAA technology in this embodiment proves that it has high sensitivity and specificity under isothermal amplification conditions. Compared with the traditional polymerase chain reaction (qPCR), RAA does not require complex temperature control equipment, and only requires 35 minutes of reaction at 39°C to obtain results, greatly shortening the detection time. This mild reaction condition and the characteristics of rapid detection can meet the needs of rapid on-site diagnosis. By using specific primers to detect BVDV and IBRV respectively, the amplification reactions of the two viruses are effectively distinguished, cross-reactions are avoided, and the accuracy of detection is improved.

[0078] The HNB colorimetric reaction further simplifies the interpretation of the results. Compared with fluorescence or electrophoresis detection methods, the HNB colorimetric reaction does not require expensive instruments and equipment, and the color change can be observed by the naked eye (positive reactions show obvious blue), which greatly reduces the technical threshold of the operation. Because the colorimetric reaction is combined with RAA amplification, the sensitivity and specificity of the test are significantly improved, ensuring that positive individuals can be accurately identified even in samples with low viral loads.

[0079] This embodiment shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention.

[0080] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV), characterized in that: The following steps are involved: Step 1: Control reaction conditions: The detection method is carried out at 39°C and the reaction time is 35 minutes; Step 2: Perform color development reaction: After the amplification reaction of the positive sample is completed, the HNB color development reaction is used to show the color change, and the test result is judged according to the color change; Step 3: For bovine viral diarrhea mucosal disease and bovine infectious rhinotracheitis virus, two pairs of specific primers are used to ensure the sensitivity and specificity of the amplification reaction; Step 4: Double verification: After RAA-HNB screening, positive samples were further verified by nucleic acid electrophoresis, where the amplified fragment sizes of infectious bovine rhinotracheitis virus were 385bp and 130bp, and the amplified fragment sizes of infectious bovine rhinotracheitis were 492bp and 359bp.

2. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 1, characterized in that: Under the reaction conditions in step 1, the recombinase, with the assistance of the single-stranded DNA binding protein, binds to the primer to form a complex, quickly recognizes and binds to the target DNA sequence, and forms a D-loop structure.

3. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 1, characterized in that: It also includes DNA polymerase to carry out chain replication, realize DNA amplification, and make the positive sample show obvious color change quickly from purple to blue, while the negative sample has no color change.

4. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV) according to claim 3, characterized in that: Two pairs of RAA primers were used for each virus to improve the amplification efficiency and specificity of the DNA chain and the sensitivity of the colorimetric detection.

5. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 4, characterized in that: The specific primers use PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids as templates, and the RAA amplification kit is used to determine the primer amplification time.

6. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 1, characterized in that: In the process of determining the reaction temperature of RAA-HNB visualization detection, PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids were used as templates, and the RAA amplification kit was used to determine the primer amplification time.

7. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV) according to claim 6, characterized in that: In the step 4, the PCR@-Blunt-BVDV-NS3 and PCR@-Blunt-BVDV-US4 positive plasmids are used as templates, and gradient dilution is used to determine the range of virus titers for visual detection.

8. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 5, characterized in that: The reaction conditions of the detection scheme optimized by RAA-HNB visualization include: reaction at 39°C for 35 minutes, observation of the color of the test tube, and verification of the results by nucleic acid electrophoresis.

9. A method for rapid detection of bovine viral diarrhea-mucosal disease (DVDV) and infectious bovine rhinotracheitis (IBRV) according to claim 1, characterized in that: It also includes the process of visualizing positive samples of BVDV and IBRV with RAA-HNB, in which previously positive animal samples are extracted and tested using the optimized RAA-HNB protocol.

10. A rapid detection method for bovine viral diarrhea-mucosal disease (DVDV) and bovine infectious rhinotracheitis (IBRV) according to claim 9, characterized in that: The detection conditions of the optimized RAA-HNB protocol were as follows: react at 39°C for 35 minutes, observe the color of the test tube, and verify the results using nucleic acid electrophoresis.

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