JCM Accepts, published online ahead of print on 21 October 2009

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J. Clin. Microbiol. doi:10.1128/JCM.00598-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development and Evaluation of One-Step TaqMan® Real-Time Reverse Transcription-PCR Assays Targeting NP, M and HA Genes of Equine Influenza Virus

Zhengchun Lu, Thomas M. Chambers, Saikat Boliar, Adam J. Branscum, Tracy L. Sturgill, Peter J. Timoney, Stephanie E. Reedy, Lynn R. Tudor, Edward J. Dubovi, Mary Lynne Vickers, Stephen Sells, and Udeni B.R. Balasuriya^*

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, Departments of Biostatistics, Statistics and Epidemiology, University of Kentucky, Lexington, KY 40546, USA; Animal Health Diagnostic Center, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 12201, USA; and Livestock Disease Diagnostic Center, University of Kentucky, KY 40511, USA

^* To whom correspondence should be addressed. Email: ubalasuriya{at}uky.edu.

The objective of this study was to develop and evaluate new TaqMan® real time RT-PCR (rRT-PCR) assays using the minor groove binding (MGB^TM) probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M) and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M and HA3) which can detect strains of H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89% and 87% sensitivity for EqFlu NP, EqFlu M and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of 10 EIV RNA molecules. Comparison of the sensitivity of rRT-PCR assays targeting the NP and M genes of both subtype with egg inoculation and the Directigen Flu A® test clearly show that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting H7 HA gene is highly specific for H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype which is thought to be extinct or possibly, still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.