A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses

Hull, Rene; Nattanmai, Seela; Kramer, Laura D.; Bernard, Kristen A.; Tavakoli, Norma P.

doi:10.1016/j.diagmicrobio.2008.07.004

« Previous Next »Diagnostic Microbiology & Infectious Disease
Volume 62, Issue 3 , Pages 272-279, November 2008

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses

Rene Hull
- Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA
Seela Nattanmai
- Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA
- Present address: Albany Medical Center, Albany, NY.
Laura D. Kramer
- Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA
- Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12222, USA
Kristen A. Bernard
- Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA
- Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12222, USA
Norma P. Tavakoli
- Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA
- Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12222, USA
- Corresponding author. Empire State Plaza, PO Box 509, Albany, NY 12201. Tel.: +1-518-474-3046; fax: +1-518-486-2693.

Received 18 April 2008; accepted 9 July 2008. published online 21 August 2008.

Abstract

A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of St. Louis encephalitis virus (SLEV) and eastern equine encephalitis virus (EEEV), for use in human and vector surveillance. The respective targets selected for the assay were the conserved NS5 and E1 genes of the 2 viruses. Because of the insufficient number of NS5 sequences from SLEV strains in the GenBank database, we determined the sequence of an approximately 1-kb region for each of 25 strains of SLEV to select primers and probes in a conserved region. Our assay has a sensitivity of 5 gene copies (gc)/reaction for EEEV and 10 gc/reaction for SLEV, and its performance is linear for at least 6 log₁₀ gc. The assay is specific and detected all strains of SLEV (69) and EEEV (12) that were tested. An internal control ensures detection of efficient nucleic acid extraction and possible PCR inhibition.

Keywords: Molecular detection, Real-time RT-PCR, Saint Louis encephalitis virus, Eastern equine encephalitis virus