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BioMed Research International
Volume 2017, Article ID 4248756, 12 pages
Research Article

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

1Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Insel Riems, Greifswald, Germany
2Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany
3Diagnostic Laboratory, Department of Clinical Sciences, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

Correspondence should be addressed to Martin Eiden; ed.ilf@nedie.nitram

Received 20 December 2016; Accepted 7 February 2017; Published 24 May 2017

Academic Editor: Satish B. Nimse

Copyright © 2017 Ariel Vina-Rodriguez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.