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Title: A real-time RT-PCR method for the universal detection and identification of flaviviruses
Authors: G. Moureau
S. Temmam
J. P. Gonzalez
R. N. Charrel
G. Grard
X. De Lamballerie
Faculte de Medecine de Marseille Universite de la Mediterranee
Mahidol University
Keywords: Immunology and Microbiology;Medicine
Issue Date: 1-Dec-2007
Citation: Vector-Borne and Zoonotic Diseases. Vol.7, No.4 (2007), 467-477
Abstract: Here we describe an optimized molecular protocol for the universal detection and identification of flaviviruses. It combines the convenient real-time polymerase chain reaction (PCR) format with a broad spectrum of flavivirus detection. This assay, based on the amplification of a 269-272 nt (depending on the flavivirus tested) region at the N terminal end of the NS5 gene, enabled the amplification of 51 flavivirus species and 3 tentative species. Sequencing of the amplicons produced by reverse transcriptase (RT)-PCR permitted the reliable taxonomic identification of flavivirus species by comparison with reference sequences available in databases, using either the BLASTN algorithm or a simple phylogenetic reconstruction. The limit of detection of the assay (2-20,500 copies/reaction depending on the virus tested) allowed the detection of different flaviviruses from a series of human sera or veterinary samples. Altogether, the characteristics of this technique make it a good candidate for the identification of previously identified flaviviruses in cell culture and the investigation of field samples, and also a promising tool for the discovery and identification of new species, including viruses distantly related to "classical" arthropod-borne flaviviruses. © 2007 Mary Ann Liebert, Inc.
ISSN: 15303667
Appears in Collections:Scopus 2006-2010

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