Simultaneous typing and HA/NA subtyping of influenza A and B viruses including the pandemic influenza A/H1N1 2009 by multiplex real-time RT-PCR

Abstract

Pandemic influenza A/H1N1 2009 and seasonal influenza viruses are currently co-circulating worldwide. A rapid, sensitive, and specific assay for distinguishing pandemic influenza A/H1N1 2009 from seasonal influenza viruses and for subtyping seasonal influenza A viruses could aid in the surveillance and control of these viral infections. Here, such a multiplex real-time RT-PCR (rRT-PCR) assay for typing influenza A and B viruses and the pandemic influenza A/H1N1 2009 is developed. This assay can also subtype seasonal influenza A viruses simultaneously. The analytical sensitivity is 10-10(4) copies/reaction. The coefficients of variation of inter-assay and intra-assay are 0.04-0.45% and 0.08-0.97%, respectively. The new multiplex rRT-PCR assay is more sensitive in subtyping seasonal influenza viruses than the conventional PCR techniques. Results obtained with this assay for the detection of pandemic influenza A/H1N1 2009 are highly consistent (96.88%) with those achieved using the US CDC's rRT-PCR protocol. A sample identified as "pandemic influenza A/H1N1 2009 positive" by the US CDC's rRT-PCR was reclassified correctly as subtype H3N2 using this assay. Taken together, this new multiplex rRT-PCR protocol could be an important tool for improving diagnosis and management of the pandemic influenza A/H1N1 2009 and seasonal influenza viruses.

Validation and Diagnostic Application of NS and HA Gene-Specific Real-Time Reverse Transcription-PCR Assays for Detection of 2009 Pandemic Influenza A (H1N1) Viruses in Clinical Specimens

“…Some cross-reactions between the influenza A (H1N1) 2009 virus and other influenza viruses have been observed by others using the HA-specific WHO reference assay (11,17). The sensitivities of the SW-NS-631, SW-H1-1076, and IA M1 assays were comparable to those described by others (2).…”

Diagnostics and Surveillance for Swine Influenza

“…Currently, HA and NA specific primers can be used for both detection and subtyping of influenza A viruses. Additionally, a number of multiplex and nested RT-PCR have been developed for subtyping with and without simultaneous detection of influenza A virus (Chander et al 2010;Fereidouni et al 2009;He et al 2009;Lam et al 2007;Li et al 2001;Stockton et al 1998;Yang et al 2010).…”

Development of a Novel Fluorophore for Real-Time Biomonitoring System

“…PCR or real-time PCR using fluorescent dyes is considered to be the most sensitive and accurate method to detect infections by influenza virus [2], [12]–[15] and malaria parasite [16]–[20]. However, the application of real-time PCR in field diagnosis has some limitations including relatively longer reaction time (1–5 h) to detect, heavier and bigger machine body making difficult to carry, and need for electric power to operate.…”