Rapid test method for inactivation of bovine viral diarrhea / mucosal disease bivalent inactivated vaccine

Abstract

The invention discloses a method for rapidly testing the inactivation of a bovine viral diarrhea / mucosal disease bivalent inactivated vaccine, and relates to the field of veterinary biological products. According to the rapid detection method disclosed by the invention, whether the virus is completely inactivated or not is monitored by detecting different cDNA (complementary deoxyribonucleic acid) gene segments of type 1 and type 2 BVDV, and the specific steps are as follows: extracting total RNA (ribonucleic acid) from the inactivated BVDV, removing DNA residues, carrying out reverse transcription, respectively carrying out PCR (polymerase chain reaction) detection aiming at the type 1 BVDV E2 gene and the type 2 BVDV non-structural protein gene, and judging whether the virus is completely inactivated or not according to an electrophoresis result. According to the method, the type 1 BVDV and the type 2 BVDV can be rapidly detected after being inactivated, whether the BVDV is completely inactivated or not can be determined within 2 hours, the operation procedure of inactivation detection in the bivalent vaccine preparation process is simplified, the detection period is shortened, the cost is saved, and the inactivation detection sensitivity and the vaccine production efficiency are improved.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and particularly relates to a rapid inactivation detection method for a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease. Background Art

[0002] Bovine viral diarrhea-mucosal disease (BVD-MD) is a disease caused by bovine viral diarrhea virus (BVDV), characterized by inflammation, erosion, necrosis of the bovine mucosa and diarrhea. This disease is globally distributed and prevalent in developed countries with cattle farming industries, and has become one of the main infectious diseases in cattle farms.

[0003] Vaccination is the most economical and effective means to prevent viral diseases. Inactivated cell vaccines have become one of the most commercially promising vaccines due to their simple preparation and stable immune effect. For bovine viral diarrhea, there are already inactivated vaccines against type 1 BVDV and multivalent inactivated vaccines combining type 1 BVDV with IBRV, BRV, etc. at home and abroad. Many domestic scholars are conducting research on subunit vaccines, which have the advantage of good safety, but the immune protection still needs to be improved. Given that both type 1 and type 2 BVDV are prevalent in China, and the cross-protection of different genotypes of BVDV is weak, there is an urgent need to develop a bivalent vaccine containing protective antigens of both genotypes 1 and 2 of BVDV.

[0004] Virus inactivation detection is a very important step in the preparation of inactivated cell vaccines. The traditional virus inactivation detection method is to blindly passage the cells for 3 generations. If the lesions are not obvious, indirect immunofluorescence assay (IFA) needs to be supplemented for detection. The whole process takes about 21 days. This method takes a long time, prolongs the vaccine production cycle, and reduces the vaccine production efficiency. Therefore, there is an urgent need to establish a rapid inactivation detection method.

[0005] Several common inactivators in veterinary inactivated cell vaccines can all destroy the nucleic acid of the virus to achieve the purpose of inactivation. During the virus proliferation process in cells, virus-related genes are continuously transcribed. Therefore, the virus mRNA can be monitored to detect whether the virus is inactivated. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for rapid inspection of inactivation of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease. After virus propagation culture and harvest of the vaccine sample, total RNA is extracted. Using the total RNA as a template, PCR amplification is carried out with primer 1 and primer 2 respectively. The PCR amplification products are subjected to agarose gel electrophoresis. If specific bands appear, it is judged as positive, indicating incomplete inactivation; if no specific bands appear, it is judged as negative, indicating complete inactivation.

[0007] The primer 1 is:

[0008] 1-F1 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’;

[0009] 1-R1 (SEQ ID NO.2): 5’-GTGGTACTATGGCCACAC-3’;

[0010] The primer 2 is:

[0011] 2-F1 (SEQ ID NO.10): 5’-TACCTCTCTATAAGGAAGGGC-3’;

[0012] 2-R1 (SEQ ID NO.11): 5’-GAAAGCGCAACCCTTCTTCATT-3’.

[0013] Furthermore, the bovine viral diarrhea virus is bovine viral diarrhea virus type 1 and / or bovine viral diarrhea virus type 2.

[0014] Furthermore, the reverse transcription system is: PrimeScript Ⅳ 1st strand cDNA Synthesis Mix 4 μl, Random 6mers 1 μl, dH2O 10 μl, and RNA template of each strain 5 μl.

[0015] Furthermore, the reverse transcription reaction program is: 30℃ for 10 min, 42℃ for 15 min, 70℃ for 15 min.

[0016] Furthermore, the PCR reaction system is: Mix 12.5 μl, H2O 8.5 μl, each upstream primer 1 μl, each downstream primer 1 μl, and cDNA template of each strain 2 μl.

[0017] Furthermore, the PCR program is: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, 30 cycles, 72℃ for 10 min.

[0018] The present invention also provides an application of the described rapid inactivation test method in the inactivation test of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease.

[0019] The present invention also provides a test kit for the inactivation test of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease, which at least comprises the following primer 1 and primer 2:

[0020] The primer 1 is:

[0021] 1-F1 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’;

[0022] 1-R1 (SEQ ID NO.2): 5’-GTGGTACTATGGCCACAC-3’;

[0023] The primer 2 is:

[0024] 2-F1 (SEQ ID NO.10): 5’-TACCTCTCTATAAGGAAGGGC-3’;

[0025] 2-R1 (SEQ ID NO.11): 5’-GAAAGCGCAACCCTTCTTCATT-3’.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The method for rapid inactivation test of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease provided by the present invention uses primers that are scientifically designed and strictly screened, and can specifically, efficiently, and sensitively perform PCR amplification on type 1 and type 2 BVDV. The method provided by the present invention takes about 3 hours, has a short test cycle, and can save a large amount of time cost compared with the traditional method. The test method provided by the present invention shortens the test cycle, saves costs, improves the sensitivity of the inactivation test, and significantly improves the vaccine production cycle and production efficiency. Description of the Drawings

[0028] Figure 1 It is an electrophoresis diagram for the screening and verification of type 1 BVDV primers. Among them, M: Marker DL2000; 1: BVDV type 1 negative control; 2: PCR amplification product of primer 1-F1 / 1-R1 for F13; 3: PCR amplification product of primer 1-F2 / 1-R2 for F13; 4: PCR amplification product of primer 1-F3 / 1-R3 for F13; 5: PCR amplification product of primer 1-F4 / 1-R4 for F13; 6: PCR amplification product of primer 1-F5 / 1-R5 for F13.

[0029] Figure 2Electrophoresis diagram for primer screening and verification of BVDV type 2. Among them, M: Marker DL2000; 1: BVDV-2 negative control; 2: PCR amplification product of primer 2-F1 / 2-R1 for FJ1; 3: PCR amplification product of primer 2-F2 / 2-R2 for FJ1; 4: PCR amplification product of primer 2-F3 / 2-R3 for FJ1; 5: PCR amplification product of primer 2-F4 / 2-R4 for FJ1; 6: PCR amplification product of primer 2-F5 / 2-R5 for FJ1.

[0030] Figure 3 Electrophoresis diagram of BVDV-1 strain F13. Among them, 1: negative control; 2: Marker; 3: positive control; 4: inactivated sample at 4 h; 5: inactivated sample at 8 h; 6: inactivated sample at 12 h; 7: inactivated sample at 16 h; 8: inactivated sample at 20 h; 9: inactivated sample at 24 h.

[0031] Figure 4 Electrophoresis diagram of BVDV-1 strain 1154. Among them, 1: inactivated sample at 24 h; 2: Marker; 3: inactivated sample at 20 h; 4: inactivated sample at 16 h; 5: inactivated sample at 12 h; 6: inactivated sample at 8 h; 7: inactivated sample at 4 h; 8: positive control; 9: negative control.

[0032] Figure 5 Electrophoresis diagram of BVDV-2 strains FJ1 and BD2. Among them, 1: Marker; 2: positive control; 3: negative control; 4: FJ1 inactivated at 4 h; 5: FJ1 inactivated at 8 h; 6: FJ1 inactivated at 12 h; 7: FJ1 inactivated at 16 h; 8: FJ1 inactivated at 20 h; 9: FJ1 inactivated at 24 h; 10: negative control; 11: positive control; 12: BD2 inactivated at 4 h; 13: BD2 inactivated at 8 h; 14: BD2 inactivated at 12 h; 15: BD2 inactivated at 16 h; 16: BD2 inactivated at 20 h; 17: BD2 inactivated at 24 h. Detailed implementation methods

[0033] The following examples are for further illustration of this patent and do not limit this patent; for the experimental methods not specifically stated in the examples, they are carried out according to conventional experimental conditions or the conditions recommended by reagent manufacturers. The reagents used in the examples, unless otherwise specified, can be purchased from commercial channels; the used bovine viral diarrhea virus type 1 (BVDV-1) strains F13 and 1154, and bovine viral diarrhea virus type 2 (BVDV-2) strain FJ1 were prepared, identified and preserved by Beijing Biomedical Technology Center of Microtek Biotechnology (Nanjing) Co., Ltd., and the bovine viral diarrhea virus type 2 (BVDV-2) strain BD2 was provided by China Agricultural University.

[0034] Example 1 Primer design

[0035] 1.1 Design of BVDV-1 Primers

[0036] Using the complete genome of the BVDV-1 strain with the NCBI accession number AJ133739.1 - NADL as a template, multiple complete genomes of type 1 BVDV were aligned using the MegAlign software to find relatively conserved regions, and primer design was performed using PrimerSelect. The following pairs of primers were designed at multiple sequence positions:

[0037] 1-F1 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’;

[0038] 1-R1 (SEQ ID NO.2): 5’-GTGGTACTATGGCCACAC-3’;

[0039] The theoretically amplified fragment of primers 1-F1 / 1-R1 is 529 bp.

[0040] 1-F2 (SEQ ID NO.3): 5’-GGCTAGCCATGCCCTTAGTAG-3’;

[0041] 1-R2 (SEQ ID NO.4): 5’-TGCAACACGTGTAGTTGGTCTT-3’;

[0042] The amplified fragment of primers 1-F2 / 1-R2: 1403 bp.

[0043] 1-F3 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’;

[0044] 1-R3 (SEQ ID NO.5): 5’-CTCCAGGTCAAACCAGTA-3’;

[0045] The amplified fragment of primers 1-F3 / 1-R3: 770 bp.

[0046] 1-F4 (SEQ ID NO.6): 5’-ATGCCATAGCAAAGGACGAA-3’;

[0047] 1-R4 (SEQ ID NO.7): 5’-TCAGCAGTAGGTACAGCAGC-3’;

[0048] The amplified fragment of primers 1-F4 / 1-R4: 1194 bp.

[0049] 1-F5 (SEQ ID NO.8): 5’-CACCTAAACTGCAAACCTGA-3’;

[0050] 1-R5 (SEQ ID NO.9): 5’-ATGACTATTAGTACAACCAT-3’;

[0051] Amplified fragment of primer 1-F5 / 1-R5: 1400 bp.

[0052] 1.2 Design of primers for BVDV-2

[0053] Using the complete genome of BVDV-2 strain with NCBI accession number AZQ00659.1 as a template, multiple complete genomes of type 2 BVDV were aligned by MegAlign software to find relatively conserved regions, and primers were designed using PrimerSelect. The following 5 pairs of primers were designed at different regional positions:

[0054] 2-F1 (SEQ ID NO.10): 5’-TACCTCTCTATAAGGAAGGGC-3’;

[0055] 2-R1 (SEQ ID NO.11): 5’-GAAAGCGCAACCCTTCTTCATT-3’;

[0056] The length of the amplified fragment of primer 2-F1 / 2-R1 is approximately 1048 bp.

[0057] 2-F2 (SEQ ID NO.12): 5’-TCGAGATGCCATGTGGACGAG-3’;

[0058] 2-R2 (SEQ ID NO.13): 5’-TCATCTTACCTTTTTCTAT-3’;

[0059] Amplified fragment of primer 2-F2 / 2-R2: 712 bp.

[0060] 2-F3 (SEQ ID NO.14): 5’-ATTCTAGCGACGAAGATGAA-3’;

[0061] 2-R3 (SEQ ID NO.15): 5’-GGTACTCATCTAAGAATATATA-3’;

[0062] Amplified fragment of primer 2-F3 / 2-R3: 1030 bp.

[0063] 2-F4 (SEQ ID NO.16): 5’-AAGTGTGAGAAGAGAGTGAGG-3’;

[0064] 2-R4 (SEQ ID NO.17): 5’-GCCAAGCTTTTGTAGAGTGCT-3’;

[0065] Amplified fragment of primer 2-F4 / 2-R4: 1040 bp.

[0066] 2-F5 (SEQ ID NO.18): 5’-ATAAGGAATGGTCAGAGGGGTA-3’;

[0067] 2-R5 (SEQ ID NO.19): 5’-CTGTCTATTTACAATATTTACA-3’;

[0068] Amplified fragment of primer 2-F5 / 2-R5: 1013 bp.

[0069] Example 2 Primer Screening and Verification

[0070] Use primers 1-F1 / 1-R1, 1-F2 / 1-R2, 1-F3 / 1-R3, 1-F4 / 1-R4, 1-F5 / 1-R5 of BVDV-1 type to amplify virus of F13 strain respectively, and use primers 2-F1 / 2-R1, 2-F2 / 2-R2, 2-F3 / 2-R3, 2-F4 / 2-R4, 2-F5 / 2-R5 of BVDV-2 type to amplify virus of FJ1 strain. The specific steps are as follows:

[0071] 2.1 Extract total RNA of virus solutions of F13 strain and FJ1 strain according to the method described in the kit (RNA extraction kit, TIANGEN, Y2130).

[0072] 2.1.1 Use a pipette to add 560 μl of Carrier RNA working solution into a clean 1.5 ml centrifuge tube;

[0073] 2.1.2 Add 140 μl of plasma / serum / lymph fluid (the sample needs to be balanced to room temperature) into the centrifuge tube. Vortex for 15 sec to mix evenly and incubate at room temperature for 10 min;

[0074] 2.1.3 Centrifuge to collect the liquid attached to the tube wall and tube cap, add 560 μl of absolute ethanol, cover the tube cap and vortex for

[15] sec; 2.1.4 Centrifuge to collect the liquid attached to the tube wall and tube cap, transfer 630 μl of the liquid in the centrifuge tube to an RNase-Free adsorption

[0075] Column CR2 (the adsorption column is placed in the collection tube), cover the tube cap, centrifuge at 6000 x g (8000 rpm) for 1 min, discard the waste liquid,

[0076] Put the adsorption column back into the collection tube;

[0077] 2.1.5 Carefully open the lid of the adsorption column, add 500 μl of buffer GD (please check whether absolute ethanol has been added before use), cover the tube cap, centrifuge at 6000 x g (8000 rpm) for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0078] 2.1.6 Carefully open the lid of the adsorption column, add 500 μl of rinsing solution RW (please check whether absolute ethanol has been added before use),

[0079] Cover the tube cap, centrifuge at 6000 x g (8000 rpm) for 1 min, discard the waste liquid, put the adsorption column back into the collection tube, and repeat this step; 2.1.7 Put the adsorption column back into the collection tube, centrifuge at 13,400 x g (12,000 rpm) for 3 min to completely dry the adsorption membrane, and discard the waste liquid;

[0081] 2.1.8 Place the adsorption column into an RNase-Free centrifuge tube (1.5 ml), carefully open the lid of the adsorption column, suspend and add 60 μl of RNase-Free ddH2O to the middle part of the adsorption membrane, cover the lid, and let it stand at room temperature for 5 min. 6000 x g

[0082] (8000 rpm) Centrifuge for 1 min, and the total RNA extracted is in the EP tube.

[0083] 2.2 Reverse transcription

[0084] Perform reverse transcription using a reverse transcription kit (TaKaRa PrimeScript IV 1st strand cDNA Synthesis Mix ALG0736A): The reverse transcription system is 4 μl of PrimeScript IV 1st strand cDNA Synthesis Mix, 1 μl of Random 6mers, 10 μl of dH2O, and 5 μl of F13 strain / FJ1 strain RNA template; The reaction program is 30 °C for 10 min, 42 °C for 15 min, and 70 °C for 15 min. Obtain the F13 strain / FJ1 strain cDNA template

[0085] 2.3 PCR

[0086] The reaction system consists of 12.5 μl of Mix, 8.5 μl of H2O, 1 μl each of upstream primers 1-F1, 1-F2, 1-F3, 1-F4, 1-F5, 2-F1, 2-F2, 2-F3, 2-F4, 2-F5, 1 μl each of downstream primers 1-R1, 1-R2, 1-R3, 1-R4, 1-R5, 2-R1, 2-R2, 2-R3, 2-R4, 2-R5, and 2 μl each of F13 strain / FJ1 strain cDNA templates.

[0087] The reaction program is 5 min at 95°C; 30 cycles of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C; and 10 min at 72°C.

[0088] 2.4 Electrophoresis

[0089] Electrophoresis was performed using a 10% agarose gel. For F13, only the 1-F1 / 1-R1 primer pair could amplify specific bands, and the results are as Figure 1 shown; for FJ1, only the 2-F1 / 2-R1 primer pair amplified specific bands, and no specific bands were observed for other primer pairs. The results are as Figure 2 shown. Based on the results, it was determined that type 1 BVDV could be identified by PCR amplification using the 1-F1 / 1-R1 primer pair, and type 2 BVDV could be identified by PCR amplification using the 2-F1 / 2-R1 primer pair.

[0090] Example 3 Inactivation test of BVDV type 1 and type 2 viruses at different times after inactivation using conventional PCR

[0091] 3.1 Inactivation

[0092] F13 and 1154 strains of BVDV type 1 and BD2 and FJ1 strains of BVDV type 2 were inactivated respectively. Samples were taken at 4, 8, 12, 16, 20, and 24 h during the inactivation process, nucleic acids were extracted respectively, reverse transcription was carried out followed by PCR, and the PCR products were electrophoresed. The electrophoresis results are as Figure 3 , Figure 4 and Figure 5 shown.

[0093] The electrophoresis results showed that no specific bands could be amplified after 12 h of inactivation for all 4 BVDV viruses.

[0094] 3.2 Sequencing analysis

[0095] The PCR products of F13 and 1154 strains of BVDV-1 type and BD2 and FJ1 strains of BVDV-2 type were sent to a sequencing company for testing. After sequence analysis, it was determined that the amplified regions of the two strains of BVDV-1 type had 99% homology with the same regions of BVDV-1 type viruses such as AJ133739.1-NADL, and the PCR amplified regions of the two strains of BVDV-2 type had 98% homology with the same regions of BVDV-2 type viruses such as AZQ00659.1. That is, the PCR products amplified with the designed BVDV-1 primers 1-F1 / 1-R1 and BVDV-2 primers 2-F1 / 2-R1 were all specific products.

[0096] Example 4 Inactivation test of samples of inactivated BVDV-1 type and 2 type viruses at different times by cell culture method 4.1 Blind passage for 2 generations

[0097] Samples of inactivated F13 and 1154 strains of BVDV-1 type and BD2 and FJ1 strains of BVDV-2 type at 4, 8, 12, 16, 20, and 24 h were respectively inoculated into 6-well plates filled with confluent monolayer MDBK cells at a volume ratio of 10%. After culturing in a 5% CO2 incubator at 37 °C for 96 h, they were frozen and thawed 3 times repeatedly, and then inoculated into MDBK cells in 6-well plates again at a volume ratio of 10%. The second-generation culture was carried out in a 5% CO2 incubator at 37 °C until 96 h, and then frozen and thawed 3 times repeatedly.

[0098] 4.2 IFA detection of the third generation

[0099] 2×10 5 cells / ml, 200 μL per well, were seeded into 96-well plates. The next day, after the cell confluence reached 70% - 80%, the virus solution of the second generation of each sample was added to the 96-well plates at a ratio of 10%. After culturing in a 5% CO2 incubator at 37 °C for 72 h, the liquid was discarded, washed once with 200 μL / well of PBS, and then fixed solution (acetone: methanol = 1:1), 100 μL per well, was added and incubated at 4 °C for 20 min - 30 min; the fixed solution was aspirated, washed twice with 200 μL / well of PBS, and after discarding the PBS for the second time, it was blotted dry with absorbent paper; BVDV monoclonal antibody diluted 1:600 with primary antibody diluent was added, 50 μL per well, and incubated at 37 °C for 1 h; the primary antibody was aspirated, washed twice with 200 μL / well of PBS, and after discarding the PBS for the second time, it was blotted dry with absorbent paper; FITC-labeled anti-mouse IgG secondary antibody diluted 1:200, 50 μL per well, was added and incubated at 37 °C in the dark for 1 h; under dark operation: the liquid was aspirated, washed twice with 200 μL / well of PBS, and after discarding the PBS for the second time, it was blotted dry with absorbent paper. After adding 100 μL of PBS to each well, the infection rate was observed and photographed under a fluorescence microscope.

[0100] The results of IFA detection were 100% consistent with those of PCR, as shown in Table 1.

[0101] Table 1 Results of detecting inactivated samples of BVDV-1 and BVDV-2 viruses by IFA and PCR

[0102]

Claims

1. A method for rapid inspection of inactivation of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease, characterized in that, After virus propagation culture and harvest of vaccine samples, total RNA is extracted. Using the total RNA as a template, PCR amplification is performed with primer 1 and primer 2 respectively. The PCR amplification products are subjected to agarose gel electrophoresis. If specific bands appear, it is judged as positive, indicating incomplete inactivation; if no specific bands appear, it is judged as negative, indicating complete inactivation. The primer 1 is as follows: 1-F1 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’; 1-R1 (SEQ ID NO.2): 5’-GTGGTACTATGGCCACAC-3’; The primer 2 is as follows: 2-F1 (SEQ ID NO.10): 5’-TACCTCTCTATAAGGAAGGGC-3’; 2-R1 (SEQ ID NO.11): 5’-GAAAGCGCAACCCTTCTTCATT-3’.

2. The method for rapid inspection of inactivation of the bovine viral diarrhea / mucosal disease bivalent inactivated vaccine according to claim 1, characterized in that The bovine viral diarrhea virus / mucosal disease is bovine viral diarrhea virus type 1 and / or bovine viral diarrhea virus type 2.

3. The method for rapid inspection of inactivation of the bovine viral diarrhea / mucosal disease bivalent inactivated vaccine according to claim 1, characterized in that, The reverse transcription system is: PrimeScriptⅣ 1st strand cDNA Synthesis Mix 4 μl, Random6mers 1 μl, dH2O 10 μl, and RNA template of each strain 5 μl.

4. The method for rapid inspection of inactivation of the bovine viral diarrhea / mucosal disease bivalent inactivated vaccine according to claim 1, characterized in that The reverse transcription reaction program is: 30°C for 10 min, 42°C for 15 min, 70°C for 15 min.

5. The method for rapid inspection of inactivation of the bovine viral diarrhea / mucosal disease bivalent inactivated vaccine according to claim 1, characterized in that, The PCR reaction system is: Mix 12.5 μl, H2O 8.5 μl, each upstream primer 1 μl, each downstream primer 1 μl, and cDNA template of each strain 2 μl.

6. The method for rapid inspection of inactivation of the bovine viral diarrhea / mucosal disease bivalent inactivated vaccine according to claim 1, characterized in that, The PCR program is: 95°C for 5 min; 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, 30 cycles, 72°C for 10 min.

7. Application of the rapid inactivation test method described in claims 1 to 6 in the inactivation test of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease.

8. A test kit for the inactivation test of a bivalent inactivated vaccine against bovine viral diarrhea / mucosal disease, which at least contains the following primer 1 and primer 2: The primer 1 is as follows: 1-F1 (SEQ ID NO.1): 5’-GACCAGTGTGGTATTCAA-3’; 1-R1 (SEQ ID NO.2): 5’-GTGGTACTATGGCCACAC-3’; The primer 2 is as follows: 2-F1 (SEQ ID NO.10): 5’-TACCTCTCTATAAGGAAGGGC-3’; 2-R1 (SEQ ID NO.11): 5’-GAAAGCGCAACCCTTCTTCATT-3’.

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