A Real-Time RT-PCR Method for the Universal Detection and Identification of Flaviviruses

Vector Borne Zoonotic Dis. Winter 2007;7(4):467-77. doi: 10.1089/vbz.2007.0206.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*

Substances

  • NS5 protein, flavivirus
  • Viral Nonstructural Proteins