A real-time RT-PCR method for the universal detection and identification of flaviviruses

• Published in: Vector borne and zoonotic diseases (Larchmont, N.Y.) Vol. 7; no. 4; p. 467
• Main Authors: Moureau, G, Temmam, S, Gonzalez, J P, Charrel, R N, Grard, G, de Lamballerie, X
• Format: Journal Article
• Language: English
• Published: United States 01.12.2007
• Subjects: Animals; Blood - virology; Culicidae - virology; Flavivirus - classification; Flavivirus - genetics; Flavivirus - isolation & purification; Humans; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sequence Analysis; Ticks - virology; Viral Nonstructural Proteins - genetics
• ISSN: 1530-3667
• DOI: 10.1089/vbz.2007.0206

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.