Establishment and Application of Heminested RT-PCR Assay for Detection of Mosquito-Borne Flavivirus — Guizhou Province, China, 2018

Abstract

Introduction: Mosquito-borne flavivirus can lead to serious infectious diseases worldwide and cause high mortality and disability. In order to strengthen epidemiological investigation of flavivirus and meet the needs of early warning of diseases, a simple, rapid, and sensitive detection method needs to be established to prevent and control mosquito-borne diseases. Methods: Using the NS5 gene of flavivirus in GenBank, 3 universal primers targeting the conserved regions were designed. The complementary DNAs (cDNAs) of Japanese encephalitis virus (JEV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) were used as the template to optimize the reaction conditions. A heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) was established to verify the sensitivity and specificity of this method and to detect field-caught mosquitoes. Results: Our results showed that this method exhibited better specificity, higher sensitivity, and the ability to detect multiple viruses simultaneously. The lowest detection limit of JEV, DENV-2, YFV, and WNV was 3×104, 3×106, 3×105, and 3×104 copies/μL, respectively. Mosquito-borne flavivirus was successfully detected in the field-caught mosquito samples using the method established in this study. Discussion: The hnRT-PCR method established in this study can be employed for the rapid detection of flavivirus and provide technical support for early and rapid diagnosis of mosquito-borne flavivirus.