Development and comparison of molecular assays for the rapid detection of the pandemic influenza A (H1N1) 2009 virus

Abstract

Human infection with the novel pandemic influenza A (H1N1) virus was first identified in April 2009. Two months later, the World Health Organization (WHO) had raised the pandemic level to phase 6. Rapid case identification is essential for prompt patient management and public health actions. This study developed real-time and conventional reverse transcription-polymerase chain reaction (rRT-PCR and cRT-PCR) assays for pandemic H1N1 detection, and compared their sensitivities with protocols developed by WHO reference centres. Altogether, three rRT-PCR and one cRT-PCR targeting the matrix gene for universal detection of influenza A; three rRT-PCR, one cRT-PCR targeting the hemagglutinin (HA) gene for specific detection of pandemic H1N1; and one multiplex cRT-PCR for differentiating co-circulating seasonal H1N1, H3N2, and pandemic H1N1 were examined. The lower detection limit ranged from 1.252 to 125.2 copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All assays showed 100% sensitivity for "optimal" specimens (nasopharyngeal samples collected within 4 days after illness onset). For the other 36 samples, cRT-PCR assays were less sensitive except that the new Protocol I-cRT-pdmH1 still retained 100% sensitivity. The new Protocol F-rRT-PCR-pdmH1 was the only pandemic virus-specific rRT-PCR assay with 100% sensitivity across all specimen categories. In conclusion, rRT-PCR assays are 10-fold more sensitive than cRT-PCR assays. The newly developed cRT-PCR assay targeting the HA gene allows rapid, specific, and sensitive screening of this novel agent, which can serve as an alternative for laboratories where a real-time PCR machine is not available.

Disproportionate impact of pandemic (H1N1) 2009 influenza on Indigenous people in the Top End of Australia's Northern Territory

“…It is possible that the actual nH1N1 admission rates were higher than estimated here. Although PCR analysis of optimal samples has a sensitivity of nearly 100%, 15 the similar demographics, outcomes and epidemic curves for non‐influenza ILI and nH1N1 raise the possibility that a proportion of the non‐influenza ILI cases were false negatives. Alternatively, other circulating respiratory viruses may have caused similar clinical presentations.…”

Comparative Pathology in Ferrets Infected with H1N1 Influenza A Viruses Isolated from Different Hosts

“…RNA was further purified using an RNeasy kit (Qiagen, Inc., Valencia, CA). One-step real-time reverse transcription-PCR (RT-PCR) for the detection of the H1N1 M gene was performed following the protocol established by the WHO Collaborating Centre for influenza at the Centers for Disease Control and Prevention (Atlanta, GA) using a One-Step RT-PCR kit (Qiagen, Inc.) (22). Controls included RNA extracted from virus stocks.…”

One-Step Multiplex Reverse-Transcriptase PCR for Detecting Pandemic (H1N1) 2009 Influenza Virus

“…The detection limits of most of the real-time RT-PCR methods for pandemic (H1N1) 2009 influenza virus have been between 1 and 200 copies [14,18,20,32], and our system's detection limit is approximately 100 copies. There is a possibility that the sensitivity of the this assay might be lower than we verified if we were to test field nasal swab samples, which include many biological substances such as blood, nasal mucosa and nasal mucus.…”