Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure
Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure
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DOI：
10.2166/wst.2011.249
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发表时间：
2011-01-01
影响因子：
2.7
通讯作者：
Ohgaki, S.
中科院分区：
文献类型：
--
作者：
Kim, K.;Katayama, H.;Ohgaki, S.
建立了一种在RT-PCR前用单叠氮乙锭(EMA)处理区分受损病毒或裸露病毒RNA与完整病毒的方法。应用的EMA处理包括三个步骤：(1)EMA剂量，(2)光照，(3)用旋转柱凝胶过滤进行额外的纯化。加入10微克/毫升的EMA和300 S的光照射，病毒核糖核酸浓度可减少约4个对数。虽然残留的EMA可以作为RT-PCR的抑制剂，但它的作用被旋转柱凝胶过滤或QIAamp(R)病毒RNA迷你试剂盒所降低。对热处理后的PV1进行EMA-RT-PCR检测。EMA-RT-PCR检测结果与PV1热灭活后的空斑检测结果基本一致。虽然这是对EMA-RT-PCR方法对RNA病毒适用性的初步研究，但结果表明，该方法可能适用于选择性地检测水中具有流行病学意义的重要肠道病毒，如肠道病毒和诺如病毒。
A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 mu g/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp (R) Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EmA-RT-PcR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.