Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR), yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito ribonucleic acid (RNA). In this paper, we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection. Our new oligo design to detect ZIKV RNA included in silico analysis of both viral and mosquito (Ae. aegypti and Ae. albopictus) genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay. Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response to virus infection. The experimental approach to detect ZIKV RNA in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

中文翻译：

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埃及伊蚊样品中ZIKV RNA检测的一步式RT-qPCR分析：研究ZIKV感染期间感染和基因表达的方案

寨卡病毒（ZIKV）在被蚊子叮咬后会传播给人类。在全球化和气候变化的情况下，在具有有效媒介的地区，暴发的频率已经并且将会增加，这表明需要不断改进媒介种群中的ZIKV检测工具。一种简单，快速，灵敏的病毒检测方法是定量逆转录聚合酶链反应（qRT-PCR），但为在哺乳动物细胞和样品中进行ZIKV检测而优化的寡核苷酸在用于蚊子核糖核酸（RNA）时反复显示出高背景。在本文中，我们提出了一种单步qRT-PCR协议，该协议可用于检测蚊子中的ZIKV并评估同一蚊子样品和RNA中的基因表达。与文献中最常用的方法相比，该方法是一种成本更低的qRT-PCR方法，并且本底更低，可以放心地进行检测。我们用于检测ZIKV RNA的新寡核苷酸设计包括对病毒和蚊子（埃及伊蚊和白纹伊蚊）基因组进行计算机分析，靶向亚洲和非洲ZIKV谱系之间保守的序列，但不匹配伊蚊的基因组。这种测定法将使研究人员避免由于病毒整合到蚊子基因组中而导致昆虫样品中的非特异性扩增，这种现象在野生和定居的蚊子种群中众所周知。使用体外转录的ZIKV RNA构建的标准曲线可优化测定的灵敏度，效率和可重复性。最后，该测定法成功地用于检测被感染蚊子中的ZIKV RNA并检测Defensin A基因的表达，该基因是埃及伊蚊对病毒感染的免疫应答中涉及的一种抗菌肽（AMP）。本文介绍的检测埃及伊蚊中ZIKV RNA的实验方法被证明是特异性，灵敏和可靠的，此外，它还可以分析ZIKV感染期间蚊子基因的表达。