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Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes

机译：一步RT-PCR检测在蚊子中黄病毒RNA检测中的应用

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The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.
机译：考察了在野外采集的蚊子中一步检测病毒RNA的RT-PCR方法的条件，以及一步RT-PCR检测被感染蚊子的保存期。我们将几种病毒RNA提取方法与带有登革热病毒（DV），日本脑炎病毒（JEV）和黄热病病毒（YFV）的人工污染蚊子池进行了比较，并确定了斑块形成单位（PFU）的数量，以建立一种条件步骤RT-PCR。在这项研究中，最有效的RNA提取方法是ISOGEN-LS提取结合离心的蚊子匀浆的上清液。使用黄病毒通用引物在10个蚊子/管（池）中进行一步RT-PCR的检出限是DV，JEV和YFV分别为10 PFU，使用种特异性引物分别在每种蚊子中分别在100个蚊子/管中为100 PFU，100 PFU使用通用引物对的PFU /管，分别使用物种特异性引物对的10 PFU /管。将感染登革热病毒的单个蚊子与99个未感染的蚊子混合，并通过一步RT-PCR进行测试。我们可以在包含99个未感染蚊子的水池中检测到单个感染蚊子。用小鼠适应的登革1型病毒株在胸腔内接种埃及伊蚊和白纹伊蚊，并在不同温度下保存30天。然后通过一步RT-PCR进行检查，以确定合适的蚊子处理方法和运输条件。蚊子自然死亡后30天内获得了阳性结果。这些结果表明，我们不仅可以检测活蚊，还可以检测死蚊检测到的黄病毒RNA，并且可以将一步RT-PCR应用于黄病毒监测的快速，特异性和高度灵敏的工具。

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