Rapid, Single-Step RT-PCR Typing of Dengue Viruses Using Five NS3 Gene Primers

J Virol Methods. 1995 Feb;51(2-3):193-200. doi: 10.1016/0166-0934(94)00104-o.

Abstract

In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Consensus Sequence
  • DNA Primers
  • Dengue / diagnosis
  • Dengue Virus / classification
  • Dengue Virus / genetics
  • Dengue Virus / isolation & purification*
  • Humans
  • Immune Sera
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA Helicases
  • RNA, Viral / analysis*
  • RNA, Viral / blood
  • Retrospective Studies
  • Sensitivity and Specificity
  • Serine Endopeptidases
  • Species Specificity
  • Transcription, Genetic
  • Viral Nonstructural Proteins / analysis*
  • Viral Nonstructural Proteins / blood
  • Viral Nonstructural Proteins / genetics

Substances

  • DNA Primers
  • Immune Sera
  • NS3 protein, flavivirus
  • RNA, Viral
  • Viral Nonstructural Proteins
  • Serine Endopeptidases
  • RNA Helicases