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J. Clin. Microbiol. doi:10.1128/JCM.02007-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Design and validation of an H5 TaqMan real-time one-step RT-PCR and confirmatory assays for detection and verification of influenza A H5 infections in humans

Centre for Infections, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT

^* To whom correspondence should be addressed. Email: joanna.ellis{at}hpa.org.uk.

	Abstract

Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all HA clades, particularly recently circulating clade 2 H5N1 viruses. In this report the development and validation of a real-time one-step TaqMan RT-PCR assay specific for the detection of influenza A H5 viruses from clades 1, 1', 2 and 3 is described. The H5 real-time assay was shown to be highly sensitive, detecting <1PFU of H5 virus from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated using influenza A viruses of different subtypes, clinical samples containing influenza A H1N1, H3N2, H5N1 and influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control, and confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to the diagnostic testing of suspect influenza A H5 cases in the UK. Influenza A H5 viruses were not detected in the cases analysed, however, influenza A H3N2 virus was detected in 57% of suspect H5 cases. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid detection of H5 infections in humans.

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