Switching gears for an influenza pandemic: validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus

Abstract

Rapid methods for the detection and confirmatory identification of pandemic influenza A virus (also known as pandemic [H1N1] 2009) are of utmost importance. In this study, a conventional reverse transcriptase PCR (RT-PCR) assay for the detection of influenza A virus and the hemagglutinin of swine lineage H1 (swH1) was designed, optimized, and validated. Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal, or throat swab specimens collected early in the outbreak (127 negative specimens, 66 specimens with pandemic [H1N1] 2009 influenza virus, 3 specimens with seasonal [H1N1] influenza A virus, and 2 specimens with seasonal [H3N2] influenza A virus). The performance characteristics of the duplex RT-PCR assay were assessed and compared to those of various detection methods: a monoplex RT-PCR assay at the National Microbiology Laboratory, a real-time RT-PCR assay using a Centers for Disease Control and Prevention protocol, an in-house multiplex RT-PCR assay (targeting influenza A virus, influenza B virus, and respiratory syncytial virus), and a rapid antigen test (the Binax Now Influenza A & B assay). The sensitivity of the duplex RT-PCR assay for influenza A virus detection was 97.2%, whereas the sensitivities were 74.6%, 71.8%, 47.8%, and 12.7% for the other four assays, respectively. The duplex RT-PCR assay was also able to identify swH1 in 94% of the cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmatory identification. Only a limited number of specimens that contained influenza A virus had amounts of virus that fell below the limit of detection of the assay with the swH1 primers. Overall, the duplex RT-PCR assay is a reliable method for the simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus and would be particularly attractive to laboratories without real-time RT-PCR capabilities.

FIG. 1.

Testing algorithms for detection of influenza A viruses. (A) Prior to the pandemic (H1N1) 2009 influenza virus outbreak, routine testing for influenza A virus (Flu A), influenza B virus (Flu B), and RSV was performed in Nova Scotia. Followed the detection of influenza A virus, huH1 and huH3 subtyping was performed. Specimens that could not be subtyped were forwarded to NML for sequence analysis. (B) Following the confirmation of cases of pandemic (H1N1) 2009 influenza virus, the testing strategies focused on screening for influenza A virus and swH1. Specimens that were weakly positive for influenza A virus or that failed subtyping were referred to NML.

FIG. 2.

The monoplex and duplex RT-PCR assays have equivalent LODs. Tenfold serial dilutions of pandemic (H1N1) 2009 influenza virus RNA were subjected to the monoplex reaction (influenza A virus or swH1) or the duplex reaction (influenza A virus and swH1). Lanes 1, 100-bp ladder; lanes 2 to 10, dilutions ranging from 10⁰ to 10⁻⁸; lane 11, reagent control.

FIG. 3.

Optimization of the duplex RT-PCR assay. (A) By using RNA concentrations 10-fold less than the LOD of swH1, a gradient RT-PCR was performed at annealing temperatures spanning 50°C to 60°C. Estimates of the temperatures achieved are as follows (the numbers for the unnumbered lanes apply to the lanes from left to right, respectively): 50.0°C (lane 2), 50.3°C (lane 3), 50.9°C (lane 4), 51.7°C (lane 5), 52.8°C (lane 6), 54.3°C (lane 7), 56.0°C (lane 8), 57.4°C (lane 9), 58.5°C (lane 10), 59.3°C (lane 11), 59.8°C (lane 12), and 60.0°C (lane 13). A 100-bp ladder is found in lanes 1 and 14. (B) The duplex RT-PCR assay was performed using annealing temperatures of 50°C, 55°C, and 60°C.

FIG. 4.

Endpoint analysis of three RT-PCR assays targeting influenza A virus. Nucleic acids extracted from 10-fold serial dilutions of pandemic (H1N1) 2009 influenza virus were subjected to the influenza A virus and swH1 duplex RT-PCR assay (A); the influenza A virus, influenza B virus, and RSV triplex RT-PCR assay (B); and a real-time RT-PCR assay targeting influenza A virus. Lanes 1, 100-bp ladder; lanes 2 to 7, dilutions ranging from 10⁰ to 10⁻⁵; lanes 8, reagent control.