A Novel Pan-FlavivirusDetection and Identification Assay Based on RT-qPCR and Microarray

Abstract

The genusFlavivirusincludes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of mostFlavivirusspecies are available, there has been also a demand for a broad-rangeFlavivirusassay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all knownFlavivirusspecies. This assay has been used as a screening and confirmation tool forFlaviviruspresence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-sixFlavivirusstrains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.