荧光定量PCR检测GⅡ型扎幌样病毒方法的建立  

ESTABLISHMENT OF A FLUORESCENCE QUANTITATIVE PCR FOR DETECTING GENOGROUP Ⅱ SAPPORO-LIKE VIRUSES

作　　者：曾爱华[1] 高冬微 禇夫江 金小宝[3] 朱家勇[3] 

机构地区：[1]广东药学院药科学院,广州510240 [2]广东检验检疫技术中心 [3]广东药学院基础学院

出　　处：《现代预防医学》2010年第2期306-308,共3页Modern Preventive Medicine

基　　金：广东省科技计划资助项目(2006B36008001);广东省农业攻关科技计划项目(2005B20401017)

摘　　要：[目的]建立贝类中GⅡ型扎幌样病毒的荧光定量PCR检测方法。[方法]通过用PEG6000对贝类中GⅡ型扎幌样病毒进行浓缩,用Trizol法提取病毒RNA,设计出针对GⅡ型扎幌样病毒保守序列的通用引物与探针,建立GⅡ型扎幌样病毒荧光定量PCR检测方法。[结果]此荧光定量PCR方法对贝类中GⅡ型扎幌样病毒检测呈现高度特异性,与诺瓦克样病毒、GⅠ型扎幌样病毒等无交叉反应,最低检出限可达102拷贝/μl,线性范围为102～106拷贝,标准曲线的相关系数为0.9993。[结论]本研究建立了贝类中GⅡ型扎幌样病毒的荧光定量PCR检测方法。[Objective] To establish a TaqMan-based real-time fluorescenc quantitative polymerase chain reaction（FQPCR） for detecting Genogroup Ⅱ（GⅡ） Sapporo-like viruses（SLVs） in shellfish.[Methods] SLVs in shellfish were concentrated by PEG6000,SLVs RNA were extracted by Trizol,the universal primers and probe were designed following large scale SLVs genome consensus analysis and subsequently a real-time FQ-PCR assay for detecting GⅡ SLVs was established.[Results] The research results showed that the assay developed in this paper possessed high accuracy and repetition for G Ⅱ SLVs detection without cross-reaction with Norwalk-like viruses and GI SLVs.The sensitivity was as low as 102 copies /μl per reaction.The assay was linear within 5-log dynamic was at the range of 102 and 106 copies.The correlation coefficient of the standard curve was 0.999 3.[Conclusion] The detection method of Genogroup Ⅱ Sapporo-like viruses has been established by real-time FQ-PCR successfully in shellfish.

关 键 词：GⅡ型扎幌样病毒 贝类 TAQMAN探针 荧光定量PCR 

分 类 号：R446[医药卫生—诊断学]