猪瘟病毒TaqMan实时荧光定量PCR的建立与应用  

DEVELOPMENT AND APPLICATION OF TAQMAN REAL-TIME RT-PCR FOR CLASSICAL SWINE FEVER VIRUS

作　　者：姚俊[1] 

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,昆明650224

出　　处：《中国动物传染病学报》2012年第6期68-76,共9页Chinese Journal of Animal Infectious Diseases

基　　金：云南省自然科学基金项目(2010ZC225)

摘　　要：为了建立一种既能检测野毒,又能检测疫苗毒的猪瘟病毒TaqMan实时荧光定量PCR方法,经过对GenBank中所有瘟病毒属成员高度保守的5'端非翻译区序列比对分析后,设计出1对TaqMan Real-time RT-PCR引物、1条TaqMan探针和3条RNA标准品制备引物。猪瘟病毒石门毒株经RNA标准品制备引物RT-PCR扩增后,再经T7 RNA聚合酶体外转录制备包含检测目的片断序列的猪瘟病毒RNA标准品。通过最佳引物、探针浓度的筛选及反应条件的优化,建立了猪瘟病毒TaqMan实时荧光定量PCR检测方法。该方法批内及批间重复试验变异系数均低于2%,特异性试验仅能检测出猪瘟强毒及疫苗毒株,最低浓度检测极限为1 X 102 copies/μL,上机检测时间少于60 min,建立的标准曲线斜率(Slope)为:-3.97,截距(Intercept)为:47.41,相关系数(R2)为:0.999779。运用该方法对3份猪瘟临床组织样品及6家企业生产的5种细胞苗及2种脾淋苗进行定量检测,结果提示:临床病料含有的病毒拷贝数差异不大,而疫苗产品每头份含有的病毒拷贝数差异较大。所建立的方法具有特异、快速、灵敏、可重复性和线性关系好的特点,不仅适合于猪瘟临床样品的早期检测,也适用于疫苗生产过程中的质控及疫苗制品的效价评估。In order to develop a TaqMan Real-time RT-PCR（qRT-PCR） assay for detection of C-strain and field strains of classical swine fever virus（CSFV）,a pair of primers,a probe for qRT-PCR and 3 primers for preparing standard RNA were designed based on highly conserved 5＇UTR sequence of pestivirus genome available in NCBI GenBank.Standard RNA that harbored the detection sequence for the TaqMan Real-time RT-PCR was also prepared using RT-PCR and in vitro transcription.Then,a TaqMan real-time fluorescence quantitative RT-PCR assay for CSFV was developed and optimized regarding reaction conditions and concentrations of primers and probe.The coefficients of variation were less than 2% for test trials performed for repeatability and reproducibility.The sensitivity of the assay was 1×102 copies/μL and test time was less than 60 min.The standard curve was established with slope,intercept and R2 of-3.97,47.41,and 0.999779,respectively.The assay was applied to quantitatively detect 3 clinical samples,2 spleen vaccines and 5 cell culture vaccines obtained from 6 commercial manufacturers.The data showed a significant difference in the number of CSFV copies per dose among 7 vaccines produced by f 6 commercial manufacturers.However,there was not a significant difference among RNA extracts from 3 clinical samples.The results showed that the TaqMan Real-time RT-PCR assay was suitable for early detection of CSFV in clinical samples and also for quality control for commercial vaccines.

关 键 词：猪瘟病毒 TaqMan实时荧光定量PCR 建立 应用 

分 类 号：S852.651[农业科学—基础兽医学]