Preliminary Study On Bovine Viral Diarrhea Virus Subtypes Detection By Two-step Multiplex Nests RT-PCR

Bovine viral diarrhea/mucosal disease (BVD/MD), called BVD in short, is caused by Bovine viral diarrhea virus. Bovine infected by BVDV shows fever,mucosal erosion ulcer,leukopenia,diarrhea,persistent infection,immune toleranc,abortion, stillbirth and deformity. As the disease was widely distributed,it caused great economic losses in the world. More than 20 provinces, cities and autonomous regions had reported BVDV in our country.BVDV detection methods mainly divided into two kinds, that was serologic testing and etiology testing, but most methods depended on the detection of the pathogens. Usually a combination of diagnosis can obtain the ideal effect. In this study, the whole genome sequences of BVDV published on Genbank were compared. And also referred to the published literatures, genomic 5'UTR, and two relatively conserved regions of non-structural protein NS5B gene were selected to design and synthesize two Condom-type RT-PCR primers which could genotype BVDV nucleic acid, respectively. The test showed that the two sets of primers were able to detect different BVDV type. According to Comprehensive comparison of the advantages of primers, primer pairs targeting NS5B area were chosen to establish a multiple nested RT-PCR method. Based on published literature, the foreign gene EGFP was selected as an internal reference and a series of EGFP primers were designed and synthesized to obtain fragments of different sizes. Proper primers were selected based on comparison test to optimize reaction conditions to achieve RT-PCR and PCR monitoring the whole process, combined with the negative control, making the test results accurate and truthful. Our results showed that the method compared to commercial kits was more sensitive and could detect a minimum 10-4TCID50 the viral nucleic acid when detected serum samples. And nucleic acid detection limit was 2.6pg, and compared to the reported RT-domestic PCR it was more sensitive detection methods. Specificity tests showed that the method could distinguish BVDV and classical swine fever virus and other symptoms similar to the distinction of cattle pathogens. This method was applied to detect 322 serum samples and 132 milk samples from four different areas of the Northeast dairy farms, and some samples were detected by the commercial antigen capture ELISA test kit. And then positive samples innoculated MDBK cells, two generations of blind passages before using RT-PCR and sequenced re-examination, concordance of the results was 100%. These results proved that the nested RT-PCR method could be used for BVDV clinical and epidemiological surveillance. Diagnosis results showed that some samples from selected cattle farms had BVDV infection. In summary, this study provided a practical, simple, fast and bovine viral diarrhea virus detection methods with relatively low cost, which would provide reliable technical support for the future prevention and control of bovine viral diarrhea virus and safety testing of biological products from cattle.