检测粪便中GGⅡ型诺如病毒Real-time RT-PCR方法的建立

Development of a TaqMan-based Real-time Reverse Transcription PCR for Detecting GenogroupⅡ Norovirus in Clinical Stool Samples

诺如病毒是世界急性胃肠炎的重要病原之一。为加强其控制，通过序列比对设计出针对GGⅡ型诺如病毒保守序列的特异性引物与探针，建立了TaqMan Real-time RT-PCR方法。结果显示，此方法对诺如病毒核酸检测高度特异，与轮状病毒、腺病毒、甲型肝炎病毒等无交叉反应，最低检出限可达10^2拷贝，线性范围为100～100拷贝，标准曲线的相关系数为-1．00。针对标准品质粒检测的批内实验变异系数为0．28％～1．63％（n=6）、批间实验为0．28％～1．05％（n=3），对同一样品分6次进行RNA提取和逆转录，其变异系数为3增8％。对212份临床腹泻标本分别用常规RT-PCR和本文建立的TaqMan Real-time PCR进行检测，发现后者检出率略高于前者。这些结果提示此研究建立的诺如病毒的TaqMan Real-time RT-PCR检测方法可用于临床腹泻粪便标本的检测。

Norovirus has been recognized as one of the major causes of gastroenteritis in the world. In order to control its epidemic, we designed the specific primers and probe following large scale norovirus genome consensus analysis and subsequently established a TaqMan based Real-time RT-PCR assay for detection of genogroup Ⅱ （GG Ⅱ ） norovirus RNA. The results showed that the assay developed in this paper possessed high specificity for norovirus nucleic acid detection and without any evident cross-reaction with other viruses, including rotavirus, adenovirus and hepatitis A virus. The sensitivity of the assay was as low as 10^2 copies per reaction. The assay was linear within 8 - log dynamic range between 10^9 and 10^2 copies. The correlation coefficient of the standard curve was - 1.00. The coefficients of variation （CV） of the Ct values of the standard plasmid were 0.28% - 1.63% （n=6）in intra-assay and 0.28% - 1.05% （n= 3）in inter-assay, respectively, while the CV of the same sample in 6 RNA extractions and reverse transcriptions was 3.88 % （n = 6）. The detection rate of the assay was a little higher but not statistically different from that of the conventional RT-PCR assay by detecting 212 stool samples. Taken together, the Real-time PCR assay successfully developed in the present study revealed its potential for norovirus diagnosis in clinical stool samples.