贝类中诺如病毒检测方法的优化

Detection method optimization for norovirus in shellfish

目的优化贝类中GⅡ型诺如病毒(No V GⅡ)的检测方法。方法用No V GⅡ人工染毒贝类样本,分别比较曲通X-100缓冲液(KNT)、Tris/甘氨酸/牛肉膏(TGBE)缓冲液、甘氨酸缓冲液、脱脂牛奶、磷酸盐缓冲液(PBS)、PBS-吐温80缓冲液和大豆蛋白缓冲液的洗脱效果以及PEG沉淀法、超滤法和膜吸附法3种浓缩方法的回收效果,并采用实时荧光RT-PCR方法进行检测,最后运用优化后的方法对诺如病毒污染的贝类各组织的病毒含量进行检测,以确定最佳检测部位。结果实时荧光RT-PCR结果表明,人工染毒后的贝类样品采用KNT缓冲液洗脱,再用PEG沉淀法浓缩,回收率最好,达18.95%;诺如病毒在贝类中的分布主要集中于进出水口、鳃和消化腺。结论贝类海产品中诺如病毒的检测建议取贝类的进出水口、鳃和消化腺,用KNT缓冲液洗脱、PEG沉淀法浓缩,实时荧光RT-PCR进行检测,以提高检测率。

Objective To optimize the detection method for G Ⅱ norovirus in shellfish. Methods Shellfish samples were artificially inoculated with No V GII. The elution efficiencies of seven elution buffers（KNT,TGBE,Glycine,Non-fat milk,PBS,PBS-tween 80 and Soy Protein） were compared,and the recovery efficiencies of three concentration methods（PEG precipitation,ultrafiltation and membrane method） were also compared. The real-time fluorescence RT-PCR method was used for detection. Finally,the optimized method was used to detect the virus content of Norovirus-contaminated shellfish tissues so as to determine the best detection site. Results The results of Real-time fluorescence RT-PCR showed that KNT combined with PEG precipitation can get the best recovery rate（18. 95%）. The distributions of norovirus in shellfish were mainly in intake/outlet,gill and digestive gland. Conclusion The intake/outlet,gill and digestive gland are the suggested parts for the detection of No V in shellfish. The combination of KNT buffer elution,PEG precipitation,real-time fluorescence RT-PCR detection can improve the detection rate.