牡蛎和粪便中GⅠ、GⅡ型诺如病毒实时聚合酶链反应检测方法的建立

Study on the use of TaqMan Real-time PCR to detect genogroup Ⅰ and Ⅱ norovirus in oysters and patients＇ stool samples

目的 建立适用于牡蛎和粪便中快速、特异、灵敏的GⅠ、GⅡ型诺如病毒（NV）定量分型诊断方法。方法 通过对GⅠ、GⅡ型NV基因组保守序列的比对分析，设计高度特异的引物和探针，建立以TaqMan探针为基础的实时聚合酶链反应方法（TaqMan Real—time RT—PCR）。结果 该方法对NV核酸检测高度特异，且GI和GⅡ型之间无交叉反应，最低检出限达10^2 copies／ul。对90份新鲜牡蛎样品和37份腹泻粪便标本分别用常规RT—PCR和笔者建立的TaqManReal—time RT—PCR进行NV检测，发现牡蛎样品中后者的检出率明显高于前者，而粪便标本中两者无明显差别。同时对阳性标本的测序分析证实结果准确可靠。结论 研究中建立的TaqMan Real—time RT—PCR方法可用于海产品标本及粪便中NV定量及分型检测，可作为应对NV胃肠炎暴发的有效诊断方法。

Objective To develop a rapid, specific and sensitive diagnostic method for quantification and typing of genogroup Ⅰ and Ⅱ norovirus in oyster shellfish and stool samples from patients who had eaten them. Methods Specific primers and probe, following large scale norovirus genome consensus analysis were designed and subsequently a TaqMan based Real-time PCR assay to detect both G Ⅰ and G Ⅱ were established. Results This method showed high specificity for norovirus nucleic acid detection, and no cross-reaction among norovirus G Ⅰ and G Ⅱ . The limit on detection of NV genomes was 10^2 copies/ul. A total of 90 oysters and 37 stool specimens with diarrhea were tested for norovirus by conventional reverse transcriptional PCR （RT-PCR） assay as well as the TaqMan Real-time PCR, respectively. The norovirus detection rate in oysters by TaqMan PCR was significantly higher than that by conventional RT-PCR, but no differences between the two PCR methods were found when detecting the stool samples. Reliability of the Real-time PCR for norovirus detection was further confirmed by DNA sequencing of the positive sarnples. Conclusion This TaqMan Real-time PCR assay was proved to be a useful method for quantification and typing for norovirus in routine monitoring of both oyster shellfish and clinical samples. This method is recommended to be an effective diagnostic method for outbreak-associated gastroenteritis due to norovirus.