实时RT-LAMP与实时荧光RT-PCR检测贝类中GⅡ型诺如病毒的研究  

Comparison of Real-Time RT-LAMP and Real-Time RT-PCR for Detection of Norovirus Genogroup Ⅱ in Shellfish

作　　者：魏海燕[1] 曾静[1] 马丹[1] 张西萌[1] 

机构地区：[1]北京出入境检验检疫局,北京100026

出　　处：《检验检疫学刊》2013年第3期49-53,共5页Journal of Inspection and Quarantine

基　　金：国家质检总局科技计划项目(2009IK171;2010IK179;2011IK187)

摘　　要：[目的]建立、评价GⅡ型诺如病毒的一步法实时RT-LAMP快速检测方法。[方法]选取GII型诺如病毒ORF1与ORF2接头处高度保守基因序列设计RT-LAMP引物,经条件优化,分析该方法的检测灵敏性和特异性,并与实时荧光RT-PCR进行比较。[结果]GⅡ型诺如病毒RT-LAMP的最佳反应条件是65℃,内外引物浓度比达1:10。该方法同其余常见食源性病毒没有交叉反应。对RNA参照品其检测低限可达10个RNA拷贝/反应,同实时荧光RT-PCR的检测水平相当;在人工感染的牡蛎和缢蛏中,RT-LAMP较实时荧光RT-PCR的检测灵敏度略低10倍。[结论]本研究所建立的RT-LAMP方法为GⅡ型诺如病毒提供了一种灵敏、快速且简便的检测方法。[Objective] To establish and estimate a one-step, real-time reverse-transcription loop-mediated isothermal amplification method for rapid detection of norovirus genogroup II （NV-GII） were established and estimated. [Method] A set of primers which specificially recognize the highly conserved region of ORF1-ORF2 junction was designed. After optimizing the reaction conditions, the sensitivity and specificity of the method were discussed and compared with real-time RT-PCR. [Results] The optimal condition for RT-LAMP assay was identified at 65~C with a 1：10 ratio of inner to outer primers. The specificity of the method was validated by the absence of any cross-reaction with other food-borne viruses. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of-10 RNA copies per assay. In spiked shellfish samples, rRT-LAMP was 10-fold less sensitive than real-time RT-PCR. [Conclusion] The developed method provides a sensitive, rapid and simple tool for NV-GII detection.

关 键 词：实时RT-LAMP 实时荧光RT-PCR 诺如病毒 

分 类 号：Q784[生物学—分子生物学] S944.4[农业科学—水产养殖]