贝类中GI、GII诺如病毒快速检测与分群方法的建立

目的 建立贝类中GI、GII诺如病毒快速检测与分群方法。 方法 对比分析6株主要流行的GI、GII诺如病毒及参考诺如病毒的基因序列,筛选保守区域引物,优化建立SYBR Green I荧光定量检测与熔解曲线快速分群方法,并对实际样品检测验证。结果 引物P289/290可同时检测GI、GII诺如病毒,SYBR Green I荧光定量检测方法在病毒浓度10³~10⁹ copies 之间呈现良好的线性关系(R²=0.993,P＜0.01),熔解曲线Tm值可区分GI和GII不同基因群的诺如病毒(F=7 507.60,P＜0.05 )。120份贝类样品阳性检出率5.83%,其中阳性结果测序鉴定与快速分群方法结论一致。 结论 该方法成本低、检测快速、分群准确,具有良好重复性,适用于贝类中GI、GII诺如病毒的快速检测与分群。

Establishment of the rapid detection and gene grouping method for genogroup I and genogroup II Noroviruses in shellfish

To establish a rapid method for the detection and classification of genogroup I(GI) and genogroup II(GII) Norovirus(NoVs) in shellfish.Gene sequence of six major prevalent GI and IIGII NoVs strains and international general reference NoVs were analyzed and one pair of primers in conservative region was selected to establish a rapid detection and gene grouping method for GI and GII NoVs in shellfish. The real samples were also tested and validated. The results showed that the P289/290 primers could simultaneously detect GI and GII NoVs at the same time, and the fluorescence quantitative detection method had a good linear relationship with virus concentration in the range of 10³~10⁹ copies (R²=0.993,P＜0.01) ,and the Tm value of melting curve could distinguish the GI and GII NoVs(F=7 507.60,P＜0.05). The positive detection rate of 120 shellfish samples was 5.83%, and the sequencing gene grouping result in the positive sample was consistent with those of rapid gene grouping in the study. The method has the benefits of low cost,quick testing,good accuracy. and good repeatability. It is suitable for rapid detection and clustering of GI and GII NoVs in shellfish.