Taqman MGB探针快速定量检测VHSV方法的研究  

Absolute quantitative real-time RT-PCR assay for rapid detection of viral hemorrhagic septicemia virus (VHSV) with Taqman MGB probe

作　　者：许建明[1] 张念之[1] 蒋一男[1] 张利峰[2] 夏春[1] 

机构地区：[1]中国农业大学动物医学院农业部预防兽医学重点实验室,北京100094 [2]北京出入境检验检疫局,北京101113

出　　处：《高技术通讯》2010年第2期208-213,共6页Chinese High Technology Letters

基　　金：国家质检总局(2007IK031)资助项目

摘　　要：为建立准确实时地定量检测病毒性出血性败血症病毒(VHSV),在VHSV-N基因保守区设计了Taqman MGB探针与引物对,随后,采用体外转录技术获得了VHSV-N基因RNA,并以此为绝对定量标准品,建立了绝对定量(AQ)检测VHSV的实时荧光RT-PCR法(AQ-RT-PCR方法),并与世界动物卫生组织(OIE)推荐的普通RT-PCR法进行了比较。此荧光RT-PCR法特异性好,与其他鱼类弹状病毒无交叉反应。检测线性范围为10^(10)～10~2拷贝/反应,灵法度达10~2拷贝/反应。此检测灵敏度比OIE推举的RT-PCR法高出5个数量级,比嵌套RT-PCR高出1个数量级。此法是出入境检疫VHSV的有效方法。An absolute quantitative （AQ） real-time RT-PCR （AQ-RT-PCR） method is established and developed for viral hemorrhagic septicemia virus （VHSV） detection. Firstly, the Taqman MGB probe and the primers are designed from highly conserved regions of nucleoprotein （N） gene of VHSV. Secondly, the AQ-RT-PCR method is established using the quantitative standard samples obtained from the in vitro transcripted VHSV N gene. The comparison of the AQ-RT-PCR with the conventional RT-PCR shows that the AQ-RT-PCR is more specific and there are no cross reactions with other fish Rhabdoviridae viruses. The linear range of the AQ-RT-PCR assay is from 10l~ copies/reaction to 100 copies/reaction. The low quantitative detection limit is 100 copies/reaction. The sensitivity of the AQ-RT-PCR is higher than the conven- tional RT-PCR by five orders of magnitude and also by one order of magnitude than the nested PCR. The AQ-RT-PCR method will be efficiently used in entr-exit detection of VHSV.

关 键 词：病毒性出血性败血症病毒(VHSV) TAQMAN MGB探针 荧光RT—PCR法 定量检测 

分 类 号：S941[农业科学—水产养殖]