贝类及其产品中甲型肝炎病毒RT-PCR检测方法的研究

    吴斌，肇慧君，王玫，袁文泽

    辽宁出入境检验检疫局技术中心，辽宁 大连 116001

    摘要：目的建立一种快速、简便、灵敏的检测贝类中甲型肝炎病毒RT-PCR方法，能够有效防止甲肝病毒的传播和对口岸进口贝类产品的监控。方法通过对病毒富集的3种方法（大体系PEG 8000、小体系PEG 6000和小体系PEG8000富集法）、提取RNA的2种方法（Trizol法和试剂盒法）以及RT-PCR检测的2种方法（一步法和两步法）进行比较，确立一种快速检测贝类中甲肝病毒的RT-PCR方法。结果经比较，采用小体系肠道样本检测比采用全贝检测的富集效果更佳，并比较了小体系PEG8000与PEG6000和大体系PEG8000对病毒富集的效果，回收率分别为13.3%、7.5%、10.0%；对HAV总RNA的2种提取方法进行了比较，结果均获得了纯度较高、完整性较好的总RNA，以此为模板采用一步法和两步法进行反转录和PCR反应，经1%琼脂糖凝胶电泳鉴定，从人工污染的阳性样本中扩增得到一段长度为192bp的特异PCR扩增产物。应用本研究建立的RT-PCR方法对大连地区85份贝类及产品进行HAV检测，结果未显阳性，说明样本中不含HAV。整体用时仅为5h。结论 本研究建立的检测HAV的RT-PCR方法灵敏特异、操作时间短，该方法可对贝类及其产品进行确实有效的HAV的检测。

    关键词：RT-PCR；甲型肝炎病毒；贝类；检测

    中图分类号： R512.6　文献标识码： B

    Studies on detection hepatitis A virus inshellfish and shellfish

    products with RT-PCR method

    WU Bin, ZHAO Hui-jun, WANG Mei, YUANWen-ze

    Liaoning Entry-exit Inspection and QuarantineBureau, Dalian, Liaoning 116001,China

    Abstract:   Objective  To establish a fast.simple,and sensitive method to detect HepatitisA virus (HAV), and to avoid HAV to spread and monitor import andexport shellfish in port. Methods   The efficacy ofmethod using polymerase chain reaction（PCR）to detect HAV incontaminated shellfish. Results   The study established akind of holistic approach that detects hepato-virus in theshellfishes.The comparison of two different methods for samplingshowed that the small system with the stomachs and digestivediverticular was more effective than the large system with wholeshellfish tissue. The effect of virus concentration from shellfishby mini-system PEG8000 and PEG6000 was compared with that bymacro-system PEG8000, and the recovery was13.3%、7.5%、10%respectively.Two methods were compared for isolation of total RNAfrom HAV.The total RNA with high purity and quality wasobtained.The RNA could be used for cDNA synthesis and polymerasechain reaction (PCR)..Furthermore,the detection of the shellfishproducts（85 samples） from Dalian area didn't displayedpositively,which implied there was no Hepatitis A Virus in them.Aspecific PCR fragments about 192bp was obtained. The whole processonly needs 5 hours. Conclusions   The established RT-PCRmethod can detect HAV from shellfish sensitively andefficiently.

    Key words:   RT-PCR;hepatitis Avirus; Shellfish； Detection