实时荧光定量PCR检测GI型诺瓦克样病毒方法的建立

Development of the real-time fluorescent quantitative PCR assay for the detection of Norwalk-like virus genogroup G1

目的建立临床粪便中GI型诺瓦克样病毒的实时荧光定量PCR检测方法。方法针对诺瓦克样病毒GI型保守序列,用序列比对软件设计特异性引物与探针,建立诺瓦克样病毒实时荧光定量PCR检测方法。并用常规RT-PCR和本文建立的实时荧光定量PCR对137份临床腹泻标本进行检测。结果该方法对诺瓦克样病毒检测准确,重复性好,标准曲线的线性范围为102～107拷贝,相关系数为0.9991。并且对临床标本的检出率显著高于普通RT-PCR。结论本研究建立的实时荧光定量PCR方法可用于检测临床腹泻粪便标本中的GI型诺瓦克样病毒,从而有效预防和控制该病毒的传染。

To develop a TaqMan based real-time fluorescent quantitative reverse transcription polymerase reaction（QZ RT-PCR）for the detection of Norwalk-like virus genogroup GI, specific primers and probes were designed according to the conserved sequences of the genogroup G1 following large scale genome consensus analysis. Subsequently, a real-time FQ RT-PCR was established, and in comparison to the routine RT-PCR assay this method was used to detect the Norwalk-like virus geno- group G1 in 137 stool specimens of clinical cases with diarrhea. The results showed that this method developed possessed high degree of accuracy and repetition for the detection of Norwalk-like virus RNA. The sensitivity of assay was as low as 102 copies per reaction. It was linear within 6-log dynamic range between 10^2 and 10^7 copies. In which the correlation coefficient of the standard curve was 0. 9991. The detection rate of this assay was much higher than that of the conventional RT-PCR assay, it is apparent that this method of assay is valuable for the detection of Norwalk-like virus in the stool specimens of patients with diarrhea.