Establishment Practical Diagnosis Method Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea is a type of B class infectious diseases of virus by theOIE location, it locate the second class infectious disease in the import and exportinspection and quarantine of China. The main clinical symptoms of BVD is fall off,the digestive tract mucosa erosion. It will lead to female abortion, stillbirth and teras,etc.Especially produce persistent infection and immune tolerance, often occurs inclinical testing targets and check out the situation, it brings to prevention in diagnosis,treatment of the disease.According to the above problem, this paper researchespractical diagnosis method as a starting point, designed to provide production practicein different conditions in the process of specific detection methods, concrete resultsare as follows.1established SYER Green fluorescence quantitative RT-PCR methodThis experiment aimed at5’UTR has designed a pair of specific primers, Thelength of the PCR product was181bp. This experiment established and optimizedSYER Green fluorescence quantitative RT-PCR method. Melting curve analysisresults appears a single piece in86.5±0.5℃,it does not appears primer dimmers andnonspecific amplification phenomenon, Through establish the Green fluorescencequantitative RT-PCR standard curve,the amplification efficiency is95.36%, thecorrelation coefficient is0.996, within the range of102¹08it has a good linearrelationship. The results of47serum test show that fluorescence RT-PCR is moreaccurate, and specificity, It can get results In4hours. SYER Green fluorescencequantitative RT-PCR method compared with TaqMan fluorescence quantitativeRT-PCR method, in addition to the strong specificity, high sensitivity, rapid reactionand so on in common, it is more economic than TaqMan methods, the advantages ofsimple operation.So this method can be widely used in all levels of entry-exit inspection andquarantine and other cases of BVDV detection, especially for persistent infection andimmune tolerance of the detection plays an important role. 2established BVDV indirect ELISA methodBVDV in biology has two type cell-cell lesion caused by type （CP） and the celllesion type （NCP）, in order to identify the CP and the NCP, this experiment hasestablished the indirect ELISA method.This experiment prepared respectively type CPBVDV OregonC24strains and NCP BVDV of Yak strains of virus, Using PEG2000to concentrate BVDV virus in order to get high purity of the antigen, then we preparethe CP, NCP high serum free, set up and optimize the reaction conditions, HRP-goatantir Rabbit IgG is1:2000, The optimal concentration of Antigen for coating were1:1000,serum specimens which were being detected was1:100effect time was30min,the best sealing liquid is5%BSA closed for1hour, best time of color is15min,this isthe best factors of ELISA reaction conditions. Specificity and repeatability test resultsproved that this method has the advantages of results are accurate, easy tooperate.Based on CP, NCP known positive serum were determined. According to CPtype with NCP type of antibody titer, infer according to the different recognition ofCP BVDV antibody concentration and the NCP, and the vaccine strains and wildstrains.3Confirmed Exaltation of Newcastle disease virus test （END） can detect bovineviral diarrhea virusSwine fever virus infect the pig kidney cells of pig, do not produce cellpathological changes, then Newcastle disease virus （NDV） infect the cell, can producecell pathological changes, this phenomenon is called（Exaltation of NDV, END） test,so t Newcastle disease virus strengthening experiment is one of the swine fever virusdetection methods.This experiment first cultured bovine kidney cells （MDBK）,BVDV Yak strainsinfect MDBK cells,then incubatored under37℃,5%CO₂constant temperature for3days, no cell pathological changes were observed through the microscope,10times ofthe plaque of NDV infects this cell, in the same conditions incubatored3days, thecells produced cell pathological changes, the result also shows that NDV has attachgreat importance to the role of bovine viral diarrhea virus.So, this experiment wasconcluded that the END can be a detection method of NCPBVDV.