猪瘟病毒荧光定量PCR的建立及对病毒体外复制动态的研究  

Proliferation dynamic of Classical swine fever virus in vitro using a newly developed fluorescent quantitative PCR assay

作　　者：王瑞宁[1,2] 赵孟孟[2] 李存法 李青梅[2] 张雨杭[3] 王丽[2] 李金磊[4] 郭军庆 张改平[2,3] WANG Rui-ning;ZHAO Meng-meng;LI Cun-fa;LI Qing-mei;ZHANG Yu-hang;WANG Li;LI Jin-lei;GUO Jun-qing;ZHANG Gai-ping(The department of veterinary medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;The Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Henan Supervision Institution for Veterinary Drugs and Feed,Zhengzhou 450008,China)

机构地区：[1]河南牧业经济学院动物医药学院,河南郑州450046 [2]河南省农业科学院动物免疫学实验室,河南郑州450002 [3]河南农业大学牧医工程学院,河南郑州450002 [4]河南省兽药饲料监察所,河南郑州450008

出　　处：《中国兽医杂志》2019年第11期32-36,I0004,共6页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500701);河南省科技攻关项目(182102110083,192102110007);河南牧业经济学院博士科研启动资金;河南牧业经济学院科研创新团队(2018KYTD18)。

摘　　要：为了了解猪瘟病毒(CSFV)在PK-15细胞上的增殖规律。根据GenBank中CSFV Shimen毒株的NS5A基因保守区域序列,设计1对特异性引物,以CSFV总RNA为反转录模板,经优化反应条件,建立了基于实时荧光定量PCR技术的CSFV检测方法,并对其进行了敏感性、特异性、重复性试验,结果显示,该方法重复性好、特异性强,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1 TCID_(50)/mL。利用所建立的方法研究CSFV在PK-15细胞上清中的增殖动态,并与TCID_(50)方法绘制的复制动态曲线进行比较。结果显示,两种方法测定的CSFV在PK-15细胞上的复制动态具有一定的平行关系,该方法为研究猪瘟病毒的致病机理及利用体外细胞培养毒生产疫苗提供了试验依据。Classical Swine Fever Virus(CSFV)can grow in the cell line PK-15,but the characterization of proliferation is still unknown.A Real-time quantitative PCR assay for detection of CSFV was developed using the specific primer designed according to the NS5A gene of CSFV.The Real-time quantitative PCR assay was established using the total RNA of CSFV as template.The method was specific and had good reliability for the detection of CSFV.The sensitivity of the assay was 101 copies/μL nucleic acid and 1TCID50 virus.This method was developed for the detection of the replication disciplinarian of CSFV in the PK-15 cell supernatant,which was compared to TCID50 assay.The results showed that the one-step growth curves of CSFV in PK-15 cells were parallel by the developed assay and TCID50 detection.

关 键 词：猪瘟病毒 荧光定量PCR PK-15细胞上清 增殖规律 TCID_(50) 

分 类 号：S852.65J1[农业科学—基础兽医学]