Establishment Of Sandwich ELISA,RT-PCR For Detection BVDV And Analysis Of E2 Genetic Variation

The Bovine Viral Diarrhea Virus (BVDV) was grown on MDBK cell lines, harvested and concentrated against polyethylene glycol as 80% cytopathic effect appeared. The hyperimmune serum of rabbit anti-BVDV was prepared by immunization in rabbit with purified BVDV. Mainly immunoglobulinG (IgG) of rabbit anti-BVDV was purificated by ammonium sulphate precipitation and caprylic acid- ammonium sulphate precipitation, the result of SDS-PAGE suggested that the caprylic acid- ammonium sulphate precipitation is one simple, efficient, rapid and low cost method for purification of IgG from serum.The sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for detecting BVDV antigen was established. Through the specificity test blocking test, cross test and duplication test we can see clearly that the method of sandwich ELISA was very specific, sensitive and stable, the assay has a promising application future.The method of sandwich ELISA was applied to detect BVDV antigen in feces of cow from Hebei province. 298 clinical specimens were detected by this method, 127 out of 298 samples were positive. The results demonstrated that BVDV-carrier rates of cow was 42.6%, which suggested the disease seriously occur in these areas.Based on the sequence alignment of the 5'untranslated region of BVDV strain, two primers were designed and synthesized. The expected 325bp was amplified from BVDV reference strain OregonC₂4V and NADL by RT-PCR using primer set Pa/Pb, whereas using the pair of primers nothing was amplified from HCLV, PRV and MDBK strain. The fragment of 325bp was divided into two parts as 111bp and 214bp by BspTI. The sensitivity of RT-PCR detection for BVDV is up to 10^-1 TCID₅0, and acquired the same results of three repeat experiments.Comparison has been made on the nucleotides of the E2 gene and their corresponding amino acid sequence, which include the 12 strains of BVDV downloaded from Genebank, Software DNAstar has been used for the purpose of analysis. It can be found in the result...