A real-time RT-PCR method for the universal detection and identification of flaviviruses.
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.
Moureau
G
G
Unité des Virus Emergents, Faculté de Médecine, Marseille, France.
Temmam
S
S
Gonzalez
J P
JP
Charrel
R N
RN
Grard
G
G
de Lamballerie
X
X
D000818:Animals; D001769:Blood / Q000821:virology; D009033:Culicidae / Q000821:virology; D005416:Flavivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017421:Sequence Analysis; D013987:Ticks / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000235:genetics*
C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins