A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus

An increase in activity of two mosquito-borne flaviviruses, West Nile virus (WNV) and Usutu virus (USUV), has been reported in Europe in recent years. The current epidemiological situation calls for RT-PCR methods that are able to detect not only the widespread lineage 1 (L1) WNV, but also lineage 2 (L2) WNV. In addition, the presence in Europe of the closely related USUV requires methods that can identify these three flaviviruses and permit an efficient and accurate differential diagnosis. Here we describe a new one-step real-time multiplex RT-PCR that detects and differentiates efficiently WNV-L1, WNV-L2 and USUV in a single reaction. The assay is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen quencher pairs. This enables the fluorescence emitted by each probe, characterized by distinct wavelengths, to be differentiated. This multiplex assay was very sensitive to all of the target viruses; in addition, there were no cross-reactions between the viruses and the assay did not react to any other phylogenetically or symptomatically related viruses. Quantitation was enabled through the use of in vitro-transcribed RNAs developed specifically for each virus as copy number standards. This new assay was validated using different types of experimental and field samples. (C) 2013 Elsevier B.V. All rights reserved.