Setting_up And Primery Application Of BVDV Fluorescence RT-PCR Detection

Bovine viral diarrhea virus (BVDV) is the member of pestivirus genus in the Flaviridae family, the virion is globular and enveloped with genome is single-strained positive-strain RNA approximately 12-13kb in length. There are two BVDV biotypes based on the presence or absence of visible cytopathic effect(CPE) in infected cell cultures:cytopathic(CP) and noncytopathic ( NCP). BVDV can infect bovine, deer, swine and other economy animals to cause astrointestinal disease, reproduces failue, respiratory diaorders, fetal malformation and persistent infections (PI).The typical BVDV genome consis of a 5'-nontranslated region(5'-NTR), a single open reading frame encoding all viral polypeptides and a 3'-nontranslated region(3'-NTR). The study show the 5'-NTR has stabilized secondary-structrue, and 5'-NTR functions as an internal ribosome entry site(IRES), it are thought to contain important signals for translation, transcription, replication. Because the squence of 5'-NTR is conservation, the detection primers are often designed according to this region.The diagnosis of BVDV inclued virus isolation, microtiter virus neutralization tests(VNT), indirect fluoresent-antibodies tests, an enzyme-linked immunosorbent assay and nucleic acid hybridization, common RT-PCR. Serology methods usually need long time and the results are not very exact and special, althought the common molecular biology method can additional some shortcome of serology, but the false position, pollution of environment and the terrible of reproducibility restricted the using.With the development of molecular biology, the technology of real-time fluorogenic quantitative-PCR (RT-FQ-PCR) has been consummated, especially the outside standard curve RT-FQ-PCR is acknowledged that it is the best exact and reproducibility method, it has been used in the field of genic expression, pathogeny detection and so on. In order to find a proper method for the detection of export-import, It is necessary to founding a rapid, sensitive, special, secure method.Bovine viral diarrhea virus strandard strain-Oregon C24V was cultured on the madin darby bovine kidney(MDBK) cell line, the regular CPE was presented in infected cell after intro cultured 72-hour. The cell became round and caryon was fixed to the borderline of cell envelope, there was a lot of vacuolus in the cytoplasm and the cells felled off from the bottle. 0.1mL 10-fold dilution Oregon C24V was dropped into the well of 96 well cell cultivationboard then placed to 37°C propagation. According to number of CPE and the dilution, TCID50 was 10"4 3 by Reed-Muench method.Based on 5'-NTR sequence of viral reference strains NADL, Now York-1, singer(Ia), Oregon C24V and so on , a pair of primers and a TaqMan probe were designed and synthesized, 94bp sequence was amplification. The probe for TaqMan was labeled with a fluorescent reporter dye(FAM) at 5' end and a quencher dye (TAMRA) at 3' end. The reaction optimal parameters of thermal each reaction systems included as followed:5 u L 25mM MgCl2, 0.5 u L 5U/uL Taq E, 1 u L 20 u M primer mixture and 1.5 u L 20 V- M TaqMan probe. The annealing and extension temperature is 60 °C. Then the sensibility t, specificity and reproducibility test were carried out, the results showed that the method could detect the minimum limit was 0.1 TCID50, and couldn't detect classical swine fever virus, bovine rotavirus and infectious bovine rhinotracheitisvirus, the results of every reproducibility test were high producibility.The method was applied to detect bovine swab samples of which included 30 from Sinping Dalong farm calf, 10 from Liaoyuan Jinchang farm grown up , 15 from Jilin Wanglong farm grown up , 20 from Xinglongshan Miaomiao diary farm calf and 25 from Jilin agricultural University attaches diary farm calf. The negative and positive control are correct, the results showed that 10 grown up and 25 calf were positive, the ratio of positive calf is higher than that of grown up. The method also detected intestine and content of diarrhea calf, the result was positive.Real-time fiuorogenicquantitative-RT-PCR assay was proved to be specificity and sensitivity and reproducibility, and can be applied to rapidly detect bovine viral diarrhea virus in short time.