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

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.