One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection
One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection
复制标题
DOI：
10.1186/s13071-020-4002-x
复制
发表时间：
2020-03-14
影响因子：
3.2
通讯作者：
Colpitts, Tonya M.
中科院分区：
文献类型：
--
作者：
Araujo, Ricardo Vieira Y.;Feitosa-Suntheimer, Fabiana;Colpitts, Tonya M.
寨卡病毒(Zika Virus，ZIKV)是通过被感染的蚊子叮咬传播给人类的。在全球化和气候变化的情况下，在有能力的媒介的地区，疫情爆发的频率已经并将会增加，这表明有必要继续改进媒介人群中的寨卡病毒检测工具。一种简单、快速和灵敏的病毒检测方法是定量逆转录聚合酶链式反应(qRT-PCR)，但用于检测哺乳动物细胞和样本中的ZIKV的寡核苷酸在用于蚊子核糖核酸(RNA)时一再显示出高背景。在本文中，我们提出了一种一步法QRT-PCR方法，该方法可以检测蚊子中的ZIKV，并评估同一蚊子样本和RNA中的基因表达。这种方法比文献中最常用的qRT-PCR方法便宜，背景低得多，可以进行可靠的检测。方法我们新的寡核苷酸设计，检测病毒和蚊子的电子分析中包含的ZIKV RNA(Ae。Aegypti和Ae.白纹伊蚊)基因组，靶向序列在亚洲和非洲ZIKV谱系之间保守，但与伊蚊基因组不匹配。这项检测将使研究人员避免由于病毒整合到蚊子基因组中而在昆虫样本中进行非特异性扩增，这种现象在野生和定居的蚊子种群中是已知的。结果用体外转录的ZIKV RNA构建的标准曲线，成功地检测了感染蚊体的ZIKV RNA，并检测了参与埃及伊蚊免疫应答的抗菌肽A基因的表达。结论本实验建立的检测埃及伊蚊ZIKV RNA的实验方法具有特异性、敏感性和可靠性，并可用于分析蚊虫感染ZIKV过程中基因的表达。
BackgroundZika 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.MethodsOur 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.ResultsFinally, 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.ConclusionsThe 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.