荧光定量RT-PCR定量检测诺如病毒方法的建立及其评价  

Detection of norovirus quantificationally by realtime RT-PCR and its evaluation

作　　者：陈传德[1] 周晓红[2,3] 莫艳玲[2] 李晖[2] 杨贵清[1] 卓菲[1] 

机构地区：[1]广东省深圳市罗湖区疾病预防控制中心,广东深圳518020 [2]广东省疾病预防控制中心,广州510300 [3]暨南大学医学院流行病学教研室,广州510632

出　　处：《中国卫生检验杂志》2010年第3期467-469,558,共4页Chinese Journal of Health Laboratory Technology

基　　金：广东省科技计划项目(2007A0300007-7);深圳市科技计划项目(200903254);深圳市罗湖区科技计划项目(2008068)

摘　　要：目的：建立荧光定量RT-PCR快速检测诺如病毒GⅠ、GⅡ的方法,以用于患者标本的检测、分型及绝对定量。方法：经过优化筛选出最佳反应体系及反应条件,建立荧光定量RT-PCR快速检测诺如病毒的方法;制作两种不同型号的荧光定量PCR仪的绝对定量标准曲线,并从灵敏度、特异性、重复性、对患者标本检测能力等方面对建立的方法进行综合评价。结果：该方法在两种不同型号的仪器上对108-101copy/μl范围内的病毒,具有良好的线性关系,最低灵敏度为101copy/μl。与容易引起腹泻症状的轮状病毒、腺病毒、星状病毒、肠道病毒均无交叉反应;诺如病毒GⅠ、GⅡ两种常见感染人类的亚型之间无交叉反应。批内及批间重复性实验的标准差在0.10～0.59、变异系数在0.43%-2.84%之间,显示该方法重复性较好。用该方法共检测腹泻患者粪便标本181份,其中33份诺如病毒阳性,随机抽取10份阳性标本测序分析,结果证实均为诺如病毒。结论：本研究建立的诺如病毒荧光定量RT-PCR检测方法,灵敏度、特异性、重复性、对患者标本的检测能力等均显示较好的结果,且能快速区分GⅠ、GⅡ两种感染人类的重要亚型,在诺如病毒的快速检测中有着实际推广意义。Objective：To establish a TaqMan-based realtime fluorescent quantitative RT-PCR assay for detection and quantitation of Norovirus GⅠand GⅡ.Methods：The reaction conditions and systems were optimized so that the assay for detection the Norovirus by Realtime RT-PCR was establish.The sensitivity,specificity,repetition were evaluated.Laboratory diagnoses of some clinical specimens of this methods were also conducted.Results：The TaqMan assay can detect as few as 1 copy/μl virus.It showed a high linear dynamic range of quantitaion between 108～101 copy/μl.There was no cross reaction with Rotavirus,Adenovirus,Astrovirus,Enterovirus.Also There was no cross reaction between Norovirus GⅠand GⅡ.The standard deviation（SD） of intra-assay and extra-assay was 0.10～0.59.And the coefficient of variation（CV） was 0.43%～2.84%.181 specimens were detected by this assay,31 of which were positive for Norovirus.10 positive specimens were selected to conduct the sequencing analysis and it was confirmed that all of them were Norovirus.Conclusion：The established TaqMan quantitative realtime RT-PCR is a specific and sensitive method for rapid detection of Norovirus.It can distinguish the Norovirus GⅠand GⅡ,which are the most important genotypes that infected to human beings.This method can be used for early laboratory diagnosis of Norovirus.

关 键 词：荧光定量RT-PCR 诺如病毒 快速检测 灵敏度 绝对定量 反应体系 重复性试验 标准曲线 特异性 反应条件 

分 类 号：R446.5[医药卫生—诊断学]