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

Abstract EN

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals.

While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay 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 known Flavivirus species.

This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies.

Twenty-six Flavivirus strains 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.