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Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) Flaviviridae pestivirus members, the virus particles are spherical, enveloped, nucleic acid for about 12 ~ 13kb single positive-strand RNA, based on the ability to make cultured cells to produce the cytopathic be divided into two biotypes: produced cytopathic and non-cytopathic type. BVDV mainly against cattle, deer and pigs, and other animals, causing intestinal diseases, reproductive disorders, respiratory diseases, fetal malformations and persistent infection. The BVDV genome contains a 5'-non-coding region (5'-NTR), encodes a large polyprotein open reading frame and 3'-non-coding region (3'-NTR). The study shows that the 5'-NTR can form a stable secondary structure and has ribosome entry site, the translation and replication of BVDV regulatory elements. BVDV 5'-NTR sequence is highly conserved, so often primers were designed according to this region, BVDV detection. Isolation BVDV detection method, and test, direct fluorescent antibody test, ELISA and nucleic acid hybridization, RT-PCR and other methods, but serological methods exist and more time-consuming, labor-intensive, low accuracy and other shortcomings, and the molecular Although the conventional methods of biology can compensate in some respects, but the false positive, environmental pollution, the repeatability is not higher was constrained by poor promotion and application, with the development of molecular biology, quantitative real-time fluorescence RT-PCR technology has become more sophisticated, especially the external standard real-time quantitative fluorescent PCR is considered to be by far the most accurate, repeatability best quantitative method, widely used in many fields of gene expression, pathogen detection. Establish a rapid, sensitive, specific, reproducible detection method is imperative to adapt to the requirements of the import and export quarantine. MDBK cells cultured bovine viral diarrhea virus the standard strain Oregon C24V test applications 72 hours there cell lesions (CPE): cell rounding, nuclear condensation to the edge, a large number of vacuoles in the cytoplasm, and shedding. The cell monolayer in the long 96-well cell culture plate were successively added a 10-fold dilution the Oregon C24V 0.1mL, recording the lesion number of holes and their dilution factor, calculated according to the Reed-Muench method appears Oregon C24V TCID 50 cultured = 10 -4.3 . To NADL, New York-Singer (Ia), Oregon C24V (Ib), Draper (Ib) and other the reference BVDV strains 5 'noncoding region as the reference sequence, the design and synthesis of real-time quantitative fluorescent RT-PCR primers and TaqMan fluorescent probe, designed to fragment size 94bp, TaqMan fluorescent probe 5 'end of the reporting fluorescent TAM' Jominy off fluorescence TAMAR. The preliminary design of the reaction parameters after the system parameter optimization results: the 25 mM the Mg 2 5 μL 5U/μL Taq enzyme 0.5μL 20μM primer mixture 1μL 20μM probe 1.5μL, annealing extension temperature is 60 ° C. Further sensitivity, reproducibility and specificity of the test, the results of this method minimum detection is to 0.1TCID 50 ; specificity test, BVDV overcast, the establishment of the positive control, detection of classical swine fever virus, cattle round-shaped virus, infectious bovine rhinotracheitis virus
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