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PEG8000、小体系PEG6000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。
Objective To establish a fast.simple,and sensitive method to detect Hepatitis A virus（HAV）,and to avoid HAV to spread and monitor import and export shellfish in port.Methods The efficacy of method using polymerase chain reaction（PCR）to detect HAV in contaminated shellfish.Results The study established a kind of holistic approach that detects hepato-virus in the shellfishes.The comparison of two different methods for sampling showed that the small system with the stomachs and digestive diverticular was more effective than the large system with whole shellfish tissue.The effect of virus concentration from shellfish by mini-system PEG8000 and PEG6000 was compared with that by macro-system PEG8000,and the recovery was13.3%、7.5%、10% respectively.Two methods were compared for isolation of total RNA from HAV.The total RNA with high purity and quality was obtained.The RNA could be used for cDNA synthesis and polymerase chain reaction（PCR）..Furthermore,the detection of the shellfish products（85 samples） from Dalian area didn＇t displayed positively,which implied there was no Hepatitis A Virus in them.A specific PCR fragments about 192bp was obtained.The whole process only needs 5 hours.Conclusions The established RT-PCR method can detect HAV from shellfish sensitively and efficiently.
出处
《中国国境卫生检疫杂志》
CAS
2011年第1期43-47,65,共6页
Chinese Journal of Frontier Health and Quarantine
基金
国家质量监督检验检疫总局科研基金项目(2008IK011)
