Reverse transcription-PCR-enzyme-linked immunosorbent assay for rapid detection and differentiation of alphavirus infections

Eryu Wang, Slobodan Paessler, Patricia V. Aguilar, Anne Sophie Carrara, Kaolin Ni, Ivorlyne P. Greene, Scott C. Weaver

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

Due to the lack of a rapid, simple, and inexpensive assay for detecting alphavirus infections, we combined a reverse transcription-PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) to identify human pathogenic alphaviruses that are endemic in the New World. By combining the sensitivity of PCR, the detection simplicity of ELISA, and the specificities of DNA probes, this method rapidly detected and differentiated closely related species and subtypes of several medically important alphaviruses. After an amplification using RT-PCR with primers targeting conserved sequences in the nonstructural protein 1 gene, sequence-specific, biotin-labeled probes targeted against Venezuelan, eastern, and western equine encephalitis or Mayaro virus genes were used for the detection of amplicons using ELISA. The assay is simple, fast, and easy to perform in an ordinary diagnostic laboratory or clinical setting. Nucleic acid derived from cell cultures infected with several alphaviruses, clinical specimens, and mosquito pools as well as frozen and paraffin-embedded animal tissues were detected and identified within 6 to 7 h in a sensitive and specific manner.

Original languageEnglish (US)
Pages (from-to)4000-4008
Number of pages9
JournalJournal of Clinical Microbiology
Volume44
Issue number11
DOIs
StatePublished - Nov 1 2006

ASJC Scopus subject areas

  • Microbiology (medical)

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