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. 2013 Apr 27;51(2):252–257. doi: 10.1007/s12275-013-2452-y

Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection

Jang-Hoon Choi 1, Mi-Seon Kim 1, Joo-Yeon Lee 1, Nam-Joo Lee 1, Donghyok Kwon 1, Min Gu Kang 1, Chun Kang 1,✉
PMCID: PMC7091309  PMID: 23625229

Abstract

Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010–2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.

Keywords: influenza, real-time RT-PCR, surveillance, diagnosis

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