| 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 |
| |
| 引用本文： | 张超,何金科,何延华,马旭升,陈创夫.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[J].中国畜牧兽医,2020,47(7):2239-2247. |
| |
| 作者姓名： | 张超 何金科 何延华 马旭升 陈创夫 |
| |
| 作者单位： | 1. 石河子大学动物科技学院, 石河子 832000;2. 石河子大学生命科学学院, 石河子 832000;3. 新疆医科大学厚博学院, 克拉玛依 834000 |
| |
| 基金项目： | 国家自然科学基金项目（U1803236、31760020） |
| |
| 摘 要： | 本研究旨在对进口胎牛血清中的牛病毒性腹泻病毒（BVDV）进行分离及鉴定。利用BVDV抗原和抗体检测试剂盒检测，提取胎牛血清中的病毒RNA，用5'-UTR巢式PCR进行扩增，PCR扩增产物连接pMD19-T进行测序分析。胎牛血清样品接种MDBK细胞，进行细胞传代培养，通过细胞分离培养、直接免疫荧光抗体检测对实验室进口胎牛血清样品进行病毒分离及鉴定，应用DNAStar对BVDV 5'-UTR、Npro与GenBank中公布的瘟病毒参考株进行多序列比对，采用Mega 6.0进行遗传进化分析。同时通过包被脱脂奶粉进行间接ELISA检测其中的BVDV抗体。结果显示，胎牛血清中BVDV抗原和抗体均为阳性，并且从胎牛血清中成功分离到一株新的牛源BVDV，命名为BVDV-GC株，该病毒株在MDBK细胞上进行增殖培养时未能引起细胞病变；5'-UTR与Npro PCR扩增为阳性，扩增产物大小均与预期相符；直接免疫荧光检测荧光信号为阳性；病毒滴度为10-3.6TCID50/0.1 mL；遗传进化分析表明，该分离株与USMARC-60779（BVDV-2）株有较近的亲缘关系，同属于BVDV-2型毒株；通过包被脱脂奶粉和商品化的ELISA试剂盒进行检测，结果表明脱脂奶粉中存在BVDV抗体。本研究从进口胎牛血清中分离出1株BVDV-2型非致细胞病变病毒，从脱脂奶粉中检测到BVDV抗体，表明进口胎牛血清和脱脂奶粉中都存在BVDV抗原和抗体污染，本研究为后续试验分析提供参考。
|
| 关 键 词： | 牛病毒性腹泻病毒(BVDV) 胎牛血清 分离 鉴定 脱脂奶粉 抗体检测 |
| 收稿时间： | 2020-01-15 |
Isolation of Bovine Viral Diarrhea Virus Type 2 in Fetal Bovine Serum and Detection of Antibodies in Skimmed Milk Powder |
| |
| 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 & Veterinary Medicine,2020,47(7):2239-2247. |
| |
| Authors: | ZHANG Chao HE Jinke HE Yanhua MA Xusheng CHEN Chuangfu |
| |
| Institution: | 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;2. College of Life Sciences, Shihezi University, Shihezi 832000, China;3. Hombo College, Xinjiang Medical University, Karamay 834000, China |
| |
| 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,NPro 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 Npro 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. |
| |
| Keywords: | bovine viral diarrhea virus (BVDV) FBS isolation identification skim milk powder antibody detection |
|
| 点击此处可从《中国畜牧兽医》浏览原始摘要信息 |
| 点击此处可从《中国畜牧兽医》下载免费的PDF全文 |
|
