一种通用的基于NSP5基因的A组轮状病毒荧光定量RT-PCR检测方法的建立  

Construction of a Real-time Fluorescent Quantitative RT-PCR for Detection of Group A Rotavirus

作　　者：曾渊君 李廷栋[2] 鄢小琼 许红梅[3] 杨晗[2] 贾连智 李毅坚[1] 罗国兴[1] 葛胜祥[2] 夏宁邵[2] 

机构地区：[1]厦门大学分子疫苗学与分子诊断学国家重点实验室,国家传染病诊断试剂与疫苗工程技术研究中心,生命科学学院,厦门361102 [2]厦门大学分子疫苗学与分子诊断学国家重点实验室,国家传染病诊断试剂与疫苗工程技术研究中心,公共卫生学院,厦门361102 [3]重庆医科大学附属儿童医院感染科,重庆404110

出　　处：《病毒学报》2017年第2期258-264,共7页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81501741),题目:轮状病毒VP8与VP5抗原区相互作用及解离机制的研究

摘　　要：建立一种通用的检测不同物种来源A组轮状病毒的逆转录实时荧光定量PCR方法(RT-qPCR)。根据轮状病毒保守的NSP5基因的保守序列设计引物和探针,建立并优化RT-qPCR检测体系;同时通过9份轮状病毒毒株和96份临床标本的检测对该方法和目前常用的基于NSP3基因的2种RT-qPCR方法进行灵敏度和检出率的比较。新建立的RT-qPCR方法特异性高,重复性好;与基于NSP3基因的方法相比:(1)在临床标本检测中,检出率无差异;(2)9株培养病毒检测中,NSP5体系可全部检出,两个NSP3对照体系NSP3-1和NSP3-2的检出率分别为78.78%(7/9)和88.89%(8/9);(3)NSP5体系与对照NSP3-1体系灵敏度相当,检测下限比NSP3-2体系低100倍。本研究建立的基于NSP5的荧光定量PCR体系灵敏度高,可检测毒株范围广,为检测不同物种来源的A组轮状病毒提供一种新的选择。We wished to establish a more universal real-time fluorescent quantitative reverse transcriptionpolymerase chain reaction（RT-PCR）method for detection of group A rotavirus.Primers and probes were designed based on the conserved genome sequence of non-structural protein 5（NSP5）,and a TaqManRTPCR was constructed.Nine strains of rotavirus and 72 clinical samples were detected using NSP5-based real-time RT-PCR and the commonly used non-structural rotavirus protein 3（NSP3）-based real-time RTPCR in parallel.The RT-PCR system based on NSP5 had good specificity and efficiency.Compared with NSP3-based real-time RT-PCR：（i）there was no significant difference between different real-time RT-PCR assays in the detection of clinical samples;（ii）NSP5-based real-time RT-PCR constructed in the present study could be used to detect more strains of cultured rotavirus;（iii）the sensitivity of NSP5-based RTPCR was equivalent to that for NSP3-1,whereas it was 100-fold higher than that for NSP3-2.The universal real-time RT-PCR that we constructed could be a new way to detect rotavirus from different species.

关 键 词：轮状病毒 实时荧光定量PCR TAQMAN NSP5 

分 类 号：R373.25[医药卫生—病原生物学]