| 孙亚萍,程民,宋士利,张心会,李蓉.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[J].Chinese journal of Epidemiology,2008,29(6):594-597 |
| 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 |
| Study on the use of TaqMan Real-time PCR to detect genogroup Ⅰ and Ⅱ norovirus in oysters and patients' stool samples |
| Received:January 07, 2008 |
| DOI：10.3321/j.issn:0254-6450.2008.06.019 |
| KeyWord: 实时聚合酶链反应|诺如病毒|基因型 |
| English Key Word: Real-time PCR|Norovirus|Genogroup |
| FundProject:国家自然科学基金资助项目(30671752) |
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| Abstract: |
| 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TaMan探针为基础的实时聚合酶链反应方法(TaMan 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102 copies/μl．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TaMan Real-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TaMan 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． |
| English Abstract: |
| 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 GⅠ and GⅡ were established. Results This method showed high specificity for norovirus nucleic acid detection, and no cross-reaction among norovirus GⅠ and GⅡ. The limit on detection of NV genomes was 102 copies/μl. 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 samples.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. |
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