A method for verifying RNA integrity in mosquito nucleic acid extraction

Arboviruses are infectious viruses transmitted by blood-sucking arthropods. Arboviral infections cause epidemics and outbreaks that pose a significant threat to public health. Common arboviral diseases include Zika, dengue, chikungunya and yellow fever virus. Due to the recent prevalence of arboviral epidemics, the public health community has increased its focus on arboviral control and molecular diagnostics. Molecular xenomonitoring is a type of molecular diagnostic that tests mosquito samples for pathogens instead of screening humans. Even during an epidemic, most mosquito pools will test negative for the viral RNA (vRNA). While the hope is that these results indicate an absence of vRNA, the possibility of negatives due to vRNA degradation cannot be ruled out. The overarching goal of this study is to develop an assay to demonstrate the integrity of mosquito RNA in mosquito nucleic acid isolations. Our rationale is that if the mosquito RNA is present and intact, then this strongly implies that the vRNA is also intact. Our goal was to design an RT-qPCR assay to detect a transcript from Aedes polynesiensis, the predominant mosquito vector in the South Pacific. To distinguish between DNA and RNA sequences, we selected a candidate gene with a large intron that separates the forward and reverse primers. The idea is that the intron would render the PCR product from DNA too large to amplify, while cDNA produced from the RNA would be short enough to be amplifiable. The gene we selected for this study is the EMC gene present in Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. RT-qPCR primers were designed to conserved regions of the gene. To further improve the assay’s specificity to Aedes polynesiensis, exon regions on the Aedes polynesiensis genome were sequenced and used as a template for primer design. This RT-qPCR assay will serve as a valuable RNA quality control assay for molecular xenomonitoring in the South Pacific