SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

Abstract

BackgroundThe monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures.ResultsWe developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes.ConclusionsBeing a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2373-4) contains supplementary material, which is available to authorized users.

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

“…To date, only five studies [19,21,24,43,47] have detected ZIKV RNA in mosquitoes using SYBR green. However, in three of these studies [24,43,47] commercial kits designed to obtain mostly viral RNA from samples were used, which will not allow for analysis of mosquito gene expression from the same sample. In the one study that used a standard extraction kit along with SYBR green, did not present any analysis about primers specificity, sensitivity, efficiency or reproducibility [19].…”

“…Molecular detection of ZIKV ribonucleic acid (RNA) in mosquitoes can be challenging due to the limited number of primers and probes published [14][15][16][17][18][19][20][21][22][23][24][25][26][27], as well as the presence of viral genetic material integrated in mosquito genomes [28,29], which can reduce the specificity for RNA detections by quantitative reverse transcription polymerase chain reaction (RT-qPCR) [30]. Most of these ZIKV oligos were optimized in mammalian cells and samples and show often-unresolvable background when used to detect infection in mosquito tissues.…”

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

“…At this time, only five studies [19,21,24,43,47] have detected ZIKV RNA in mosquitoes using SYBR green. However, in three of these studies [24,43,47] commercial kits designed to obtain mostly viral RNA from samples were used, which will not allow for analysis of mosquito gene expression from the same sample. In the one study that used a standard extraction kit along with SYBR green did not present any analysis about primers specificity, sensitivity, efficiency or reproducibility [19].…”

Relationship between microRNA-206 plasma levels with the severity of coronary artery conflicts in patients with coronary artery disease

“…Over the past few years, researchers have been focusing on the role of microRNAs in cellular, molecular, and various disorders and diseases (17,18).…”