Detection of West Nile virus in mosquitoes by RT-PCR

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing 'TaqMan' detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evalua ted with the following viruses: eastern equine encephalitis, Ilheus, West N ile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in in fected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 5 0 repeatedly. When the RT-PCR was used with coded samples of intrathoracica lly-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus i n infected mosquitoes or cell culture. (C) 2001 Academic Press.