诺瓦克样病毒实时荧光定量PCR检测方法研究

目的建立贝类中GⅡ型诺瓦克样病毒的实时荧光定量PCR检测方法。方法针对GⅡ型诺瓦克样病毒保守序列,设计出简并引物与探针,建立GⅡ型诺瓦克样病毒实时荧光定量PCR检测方法。并用该方法和常规PCR法对127份贝类标本进行检测。结果研究方法对GⅡ型诺瓦克样病毒检测准确和重复性好,灵敏度为10拷贝。标准曲线的线性范围为101~105拷贝,相关系数为0.9986,并且对贝类标本的检出率显著高于常规PCR。结论本研究建立了GⅡ型诺瓦克样病毒的实时荧光定量PCR检测方法,可用于贝类中GⅡ型诺瓦克样病毒污染状况及其突发事件的快速定量检测。

Development of a real-time fluorescence quantitative PCR for detecting Genogroup Ⅱ Norwalk-like viruses

Objective To develop a real-time fluorescence quantitative polymerase chain reaction（FQ-PCR）for detecting Genogroup Ⅱ （G Ⅱ ） Norwalk-like viruses（NLVs）in shellfish. Methods The degenerate primers and probe were designed following large scale NLVs genome consensus analysis and subsequently, a real-time FQ-PCR assay for detecting G Ⅱ NLVs was established. There 127 shellfish samples were detected by conventional PCR and real-time FQ-PCR respectively. Results The assay developed in this paper possessed high accuracy and reproducibility for G Ⅱ NLVs detection,The sensitivity of the assay was as low as 101 copies. The linear range was within 5-log dynamic range between 101 copies and 105copies. The coefficient of the standard curve was 0.9986. The detection rate of the assay was much higher significantly than that of the conventional PCR assay. Conclusion The detection method of GⅡ NLVs has been established by real-time FQ-PCR,it can be used for the rapid detection of G Ⅱ NLVs pollution source in shellfish and emergency of G ⅡNLVs.