基于同源加尾系统的4种腹泻病毒的多重RT-PCR检测方法  

HAND system-based four multiplex RT-PCR for simultaneous detection of four diarrhea viruses

作　　者：滕勇勇[1] 莫秋华[2] 王琪[2] 唐明慧[2] 赵德坚[1] 谭华[2] 涂承宁[2] 杨泽[2] 陈清[1] 孙虹[3,2] 

机构地区：[1]南方医科大学公共卫生与热带医学学院流行痛学系,广东广州510515 [2]珠海出入境检验检疫局,广东珠海519020 [3]南方医科大学公共卫生与热带医学学院流行病学系,广东广州510515

出　　处：《南方医科大学学报》2013年第5期724-727,共4页Journal of Southern Medical University

基　　金：国家传染病重大专项传染病监测技术平台项目(2009ZX10602-02);珠海市科技计划项目(PC20081002);珠海检验检疫局科技计划项目(ZH2009-4)~~

摘　　要：目的针对轮状病毒、诺如病毒、星状病毒和扎如病毒四种常见腹泻病毒,建立基于同源加尾(Homo-Tag AssistedNon-Dimer,HAND)系统的一步法四重RT-PCR检测方法。方法根据4种病毒基因组保守序列设计引物并在5'端加上同源尾巴序列。通过优化通用尾巴引物和特异性加尾引物的浓度等PCR参数构建四重RT-PCR反应体系,并系统评价其稳定性、特异性和灵敏度。结果成功建立基于HAND系统的4种腹泻病毒一步法多重RT-PCR检测方法。特异性分析显示四种病毒间无交叉反应,灵敏度分析显示轮状病毒、诺如病毒、星状病毒和扎如病毒的检测下限分别达到48、9.6、1.92和48 pg。结论所建立的基于HAND系统的4种腹泻病毒多重RT-PCR检测方法简便快速、灵敏特异稳定、成本低廉,非常适用于基层医学实验室。Objective To establish a one-step four multiplex reverse transcription polymerase chain reaction （RT-PCR） method based on Homo-Tag Assisted Non-Dimer System （HAND） system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus. Methods Primers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5＇ end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically. Results The 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively. Conclusion The established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

关 键 词：多重RT-PCR HAND系统 轮状病毒 诺如病毒 星状病毒 扎如病毒 

分 类 号：R440[医药卫生—诊断学]