Comparison of a Multiplex Real-Time PCR Assay with a Multiplex Luminex Assay for Influenza Virus Detection

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Abstract
We describe the development of a multiplex reverse transcription-PCR (RT-PCR) with Luminex microarray hybridization for detection of influenza virus subtypes (FLULUM). Performance of FLULUM was evaluated by comparing it to our real-time RT-PCR influenza virus assay on samples collected during two influenza seasons. Both assays targeted the matrix genes of influenza virus A (FluA M) and influenza virus B (FluB M) and the hemagglutinin genes of seasonal H3N2 (H3) and H1N1 (H1) and 2009 pandemic H1N1 (2009 H1). We evaluated FLULUM on both the Luminex LX200 and the Luminex MagPix instruments. Compared to real-time PCR, FLULUM tested on 259 specimens submitted in the 2010-2011 season showed sensitivities of 97.3% for FluA M, 90.5% for 2009 H1, 96.9% for H3, and 88.9% for FluB M. No specimens were positive for seasonal H1. FLULUM tested on 806 specimens submitted in the 2011-2012 season showed a sensitivity of 100% for FluA M, 89.9% for 2009 H1, 96.4% for H3, and 95.6% for FluB M. No cross-reactivity was observed for other respiratory viruses. Analytical sensitivity was assessed by testing dilutions of specimens with high viral loads. The limits of detection of FLULUM were comparable to those of the real-time PCR assay for FluA M, FluB M, and H3. The limits of detection for seasonal H1 and 2009 H1 were 10-fold higher for the FLULUM assay compared to real-time PCR. The FLULUM is an economic assay with high clinical sensitivity and specificity. It is particularly suited to high-volume detection of influenza viruses.

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