人星状病毒、肠道腺病毒双重实时荧光RT-PCR方法的建立及应用  

Development and Application of a Duplex Fluorescence Quantitative RT-PCR Method for Simultaneous Detection and Differentiation of Human Astroviruses and Enteric Adenoviruses

作　　者：毛彤瑶 刘诗琪 裴银辉 王萌璇 章青[2] 王宏[2] 孔翔羽[2] 李慧莹[2] 张佳艳[3] 段招军[2] 李丹地[2] MAO Tongyao;LIU Shiqi;PEI Yinhui;W ANG Mengxuan;ZHANG Qing;WANG Hong;KONG Xiangyu;LI Huiying;ZHANG Jiayan;DUAN Zhaojun;LI Dandi(School of Basie Scicences,North China University of Sciences and Technology,Tangshan 063210,China;Key Laboratory of Medical Vinuses and Viral Diseases,National Health Commission,National Institute for Viral Disease Control and Prevention,Chinese Center for Diseases Control and Prevention,Beijing 102206,China;Lushan College,Guangri University of Science and Technology,Liuchou 545616.China)

机构地区：[1]华北理工大学基础医学院,唐山063210 [2]国家卫生健康委员会医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,北京102206 [3]广西科技大学鹿山学院,柳州545616

出　　处：《病毒学报》2020年第5期855-864,共10页Chinese Journal of Virology

基　　金：国家科技重大专项(项目号:2018ZX10711-001),题目:艾滋病和病毒性肝炎等重大传染病防治;国家自然科学基金(项目号:8160813),题目:P[6]基因型轮状病毒跨物种传播的分子机制研究。

摘　　要：人星状病毒(Human astrovirus,HAstV)、肠道腺病毒(Enteric adenovirus,EAdV)是引起急性胃肠炎的两种常见病原体,目前尚未实现这两种病原体荧光定量RT-PCR的单管双重检测。为建立一种灵敏特异的双重荧光定量RT-PCR检测方法并应用于实验室样本检测,本研究选择HAstV、EAdV特异性引物和探针,优化反应体系和反应条件,并对该方法的灵敏性、特异性和稳定性进行评价。结果显示,该方法针对HAstV和EAdV的检出限分别可以达到522拷贝/μL和53.5拷贝/μL;与多种常见和罕见的人腹泻病毒没有交叉反应;批内和批间等重复性实验变异系数均小于5%;灵敏度高于常规PCR。本研究提示,建立的HAstV、EAdV双重荧光定量RT-PCR检测方法具有较好的灵敏度、特异性和稳定性,可用于实验室HAstV和EAdV的快速筛查。Human astrovirus(HAstV) and enteric adenovirus(EAdV) are two common pathogens causing acute gastroenteritis,however,single tube RT-PCR detection of these two pathogens has not been realized. We wished to develop a duplex fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR)method for simultaneous detection and differentiation of human astroviruses and enteric adenoviruses for application in clinical testing. We selected specific primers and probes of human astroviruses and enteric adenoviruses to optimize the reaction system and conditions,as well as to evaluate the sensitivity,specificity and stability of the method. The limit of detection of our method for human astroviruses and enteric adenoviruses was 522 copies/μL and 53.5 copies/μL,respectively. There was no cross-reaction with other common or rare human diarrhea-causing viruses. The coefficient of variation was <5% within assays and between assays. Our duplex fluorescence quantitative RT-PCR method was highly sensitive,specific,and stable for detection of human astroviruses and enteric adenoviruses,and could be used in patients with acute gastroenteritis.

关 键 词：人星状病毒(HAstV) 肠道腺病毒(EAdV) 急性胃肠炎 荧光定量RT-PCR 

分 类 号：R373.2[医药卫生—病原生物学] R511[医药卫生—基础医学]