胎牛血清中牛病毒性腹泻病毒2型毒株分离及脱脂奶粉中抗体的检测

doi:10.16431/j.cnki.1671-7236.2020.07.030

Abstract

Abstract: This study aimed to isolate and identify the bovine viral diarrhea virus (BVDV) in imported fetal bovine serum.The virus RNA in fetal bovine serum was extracted and amplified by 5'-UTR nested PCR.The PCR products were linked to pMD19-T for sequencing.The fetal bovine serum samples were inoculated with MDBK cells for cell subculture.The sample was identificated by cell culture,direct immunofluorescent assay,nucleotide sequencing and bioinformatics analysis.DNAStar was used to compare the multiple sequences of BVDV 5'-UTR,N^Pro and BLAST virus reference strains published in GenBank,and Mega 6.0 was used for genetic evolution analysis.At the same time,the antibody of BVDV was detected by indirect ELISA with skimmed milk powder.The results showed that BVDV antigen and antibody were both positive in fetal bovine serum,and a new bovine BVDV strain was successfully isolated from fetal bovine serum,and was named BVDV-GC strain,which failed to cause cytopathic changes in MDBK cell proliferation.The results showed that the virus strain did not cause cytopathic changes when it was cultured on MDBK cells.The PCR amplification of 5'-UTR and N^pro was positive,and the size of the amplification products was consistent with the expectation.The direct immunofluorescence was positive.The virus titer was 10^-3.6TCID₅₀/0.1 mL.Genetic evolution analysis showed that the isolate was closely related to USMARC-60779 (BVDV-2) strain,the same strain belonged to BVDV-2.The test was carried out with skimmed milk powder and commercial ELISA kit.The results showed that there was BVDV antibody in skimmed milk powder.In this study,a BVDV-2 virus was isolated from BVDV fetal bovine serum,which showed that BVDV antigen and antibody contamination existed in both imported fetal bovine serum and skimmed milk powder,providing a reference factor for the follow-up cell research.

Key words: bovine viral diarrhea virus (BVDV); FBS; isolation; identification; skim milk powder; antibody detection

CLC Number:

S852.65

ZHANG Chao, HE Jinke, HE Yanhua, MA Xusheng, CHEN Chuangfu. Isolation of Bovine Viral Diarrhea Virus Type 2 in Fetal Bovine Serum and Detection of Antibodies in Skimmed Milk Powder[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2239-2247.

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Isolation of Bovine Viral Diarrhea Virus Type 2 in Fetal Bovine Serum and Detection of Antibodies in Skimmed Milk Powder

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