| 应用荧光定量RT-PCR法检测牡蛎中诺如病毒 |
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| 引用本文： | 邓丽丽,刘巍,莫建光,谭冬梅,谢镇国,唐振柱,董柏青.应用荧光定量RT-PCR法检测牡蛎中诺如病毒[J].中国热带医学,2011,11(2):133-135. |
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| 作者姓名： | 邓丽丽 刘巍 莫建光 谭冬梅 谢镇国 唐振柱 董柏青 |
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| 作者单位： | 1. 广西壮族自治区疾病预防控制中心,广西南宁,530028
2. 广西测试分析研究中心,广西南宁,530022 |
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| 基金项目： | 广西壮族自治区科学技术厅项目，广西壮族自治区卫生厅科研课题 |
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| 摘 要： | 目的建立牡蛎样品中的诺如病毒检测方法。方法优化甘氨酸缓冲液处理牡蛎匀浆液,摸索PEG沉淀浓缩病毒的浓度,提取病毒RNA,采用荧光定量RT-PCR方法进行诺如病毒检测。应用优化后的方法检测105份牡蛎样本,并用核苷酸序列测定法对阳性PCR产物进行验证。结果甘氨酸缓冲液pH值为4.5,PEG沉淀浓度为16%时的诺如病毒富集提取效果最好,该方法的诺如病毒检出限为1.17×102个质粒拷贝。应用该方法对未知样本进行检测阳性率达14.29%,所得阳性PCR产物经核苷酸序列测序证实为诺如病毒。结论本研究方法可以应用于牡蛎中的诺如病毒检测,南宁市海鲜市场上的部分牡蛎含有诺如病毒。
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| 关 键 词： | 诺如病毒 牡蛎 富集 荧光定量RT-PCR |
Detection of norovirus in oyster by real time RT-PCR |
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| Institution: | DENG Li-li,LIU Wei,MO Jian-guang,et al.(Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention,Nanning 530028,Guangxi,P.R.China) |
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| Abstract: | Aim To develop an assay for detection of norovirus in oysters.Methods The viral RNA was extracted and purified by using an optimized protocol of virus elution with glysine buffer and virus precipitation with polyethylene glycol（PEG）.The RNA extracts were analyzed for norovirus using realtime RT-PCR.Positive PCR products were sequenced to further confirm the presence of virus.Results When the pH of glysine buffer was 4.5 and the concentration of PEG was 16%,higher levels of norovirus could be detected,with the detection limit of 1.17×102 copies.By this protocol,15（14.29%） out of the 105 samples were tested positive.All the positive PCR products were confirmed as norovirus by sequence analysis.Conclusion The method adopted in this study is applicable for use in screening norovirus in oysters.The result shows that norovirus is present in oysters from aquatic markets in Nanning.8 |
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| Keywords: | Norovirus Oyster Accumulation Real time RT-PCR |
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