贝类GⅠ型扎幌样病毒荧光定量PCR检测

Development of fluorescence quantitative PCR for detection of genogroup Ⅰ Sapporo-like viruses in shellfish

目的建立贝类中GⅠ型扎幌样病毒的荧光定量PCR检测方法。方法通过用聚乙二醇6000（PEG6000）对贝类中扎幌样病毒进行浓缩,用序列比对软件设计出针对GⅠ型扎幌样病毒保守序列的通用引物与探针,建立GⅠ型扎幌样病毒荧光定量PCR检测方法。结果浓度为10^6-10^4,10^3,10^2-10^1拷贝/μL的GⅠ扎幌样病毒检出率分别为3/3,1/3,0/3,因此,常规逆转聚合酶链反应（RT-PCR）最低检出限为10^3拷贝/μL。此荧光定量PCR方法对贝类中GⅠ型扎幌样病毒检测高度特异,最低检出限可达10^2拷贝/μL,比常规RT-PCR的低1个数量级,即灵敏度高10倍,线性范围为10^2-10^6拷贝/μL,标准曲线的相关系数为0.998 8。结论本研究建立了GⅠ型扎幌样病毒的荧光定量PCR检测方法,可用于贝类中GⅠ型扎幌样病毒污染状况及其突发事件的快速定量检测。

Objective To develop a fluorescence quantitative polymerase chain reaction（FQ-PCR） for the detection of genogroup I （GI） Sapporo-like viruses （SLVs） in shellfish. Methods SLVs were concentrated by PEG6000. The degenerate primers and probe were designed following large scale SLVs genome consensus analysis and subsequently a FQ- PCR assay for detection of GI SLVs was established. Results Tthe assay developed possessed high accuracy and repetition for SLVs detection. The sensitivity of the assay was as low as 10^2 copies per reaction. The assay was linear within 5-log dynamic range between 10^2 copies and 10^6copies. The correlation coefficient of the standard curve was 0. 9988. Conclusion The detection method of genogroup I Sapporo-like viruses was established with FQ-PCR and can be used for rapid detec- tion of SLVs pollution in shellfish and emergency of SLVs.