Diagnostic approach for the differentiation of the pandemic influenza A(H1N1)v virus from recent human influenza viruses by real-time PCR

Abstract

Background: The current spread of pandemic influenza A(H1N1)v virus necessitates an intensified surveillance of influenza virus infections worldwide. So far, in many laboratories routine diagnostics were limited to generic influenza virus detection only. To provide interested laboratories with real-time PCR assays for type and subtype identification, we present a bundle of PCR assays with which any human influenza A and B virus can be easily identified, including assays for the detection of the pandemic A(H1N1)v virus.

Principal findings: The assays show optimal performance characteristics in their validation on plasmids containing the respective assay target sequences. All assays have furthermore been applied to several thousand clinical samples since 2007 (assays for seasonal influenza) and April 2009 (pandemic influenza assays), respectively, and showed excellent results also on clinical material.

Conclusions: We consider the presented assays to be well suited for the detection and subtyping of circulating influenza viruses.

Figure 1. Real-time PCR diagnostic scheme.

Samples are spiked with FCV after arrival in the lab. After nucleic acid extraction and cDNA synthesis, all samples are examined by real-time PCR with the AM/AMsw + FCV duplex PCR assay and the BM assay. All influenza A-positive samples then undergo subtyping by the H1, H3 and H1v assays. The subtype information can be completed by the application of the corresponding N1, N2 and N1v assays. FCV-negative samples are considered non-analyzable in case that no influenza A or B virus is amplified, and are re-analyzed. Otherwise, samples are considered positive for the detected virus.