草莓中甲型肝炎病毒检测

目的:建立草莓中甲型肝炎病毒(hapatitis A virus,HAV)的有效富集方法及病毒RNA提取方法,用于草莓中HAV检测。方法:利用甲肝减毒疫苗对已知阴性的草莓样品进行人工污染,通过有效富集条件的优化及RNA提取后,采用实时荧光逆转录-聚合酶链式反应进行检测。结果:病毒富集选择Tris-甘氨酸-1 g/100 m L牛肉浸提物缓冲液洗脱、果胶酶30 U、氯仿-正丁醇为抑制剂去除剂、聚乙二醇沉淀、5℃孵育1 h等优化条件,检测灵敏度较高;最优RNA提取方法为美国ABI公司生产的RNA提取试剂盒。采用优化后的方法对草莓样品中HAV病毒的检测显示,该病毒粒子的检测灵敏度可以达31.36 CCID50/20 g样品。同时对50份送检样品进行检测,结果均为阴性。结论:所建立的病毒富集方法和核酸提取方法更适合草莓样品中HAV的检测,灵敏度较高。

An Optimized Method for the Detection of Hapatitis A Virus (HAV) in Strawberry

Objective: To establish the optimal enrichment and RNA extraction procedures for detecting hepatitis A virus
(HAV) in strawberries. Methods: After artificial contamination of known negative samples, HAV enrichment under
optimized conditions and RNA extraction were performed for analysis by real-time fluorescence PCR. Results: The virus
was enriched by elution with Tris glycine 1 g/100 mL beef extract buffer (TGBE), removal of the inhibitors using 30 U of
pectinase and a mixture of chloroform with n-butanol, precipitation with PEG, and 1 h incubation at 5 ℃. The detection
method was sensitive. The best RNA extraction results were achieved with a RNA extraction kit from ABI. The
sensitivity of the optimized method for detecting HAV particles was 31.36 CCID50/20 g of sample. Negative results
were reported for all 50 samples tested. Conclusion: The established method is suitable for the detection of HAV in
strawberry samples with high sensitivity.