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Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness

Elektra Szewczuk1, Kiran Thapa2, Terry Anninos4, Kenneth McPhie3, Geoff Higgins4, Dominic E Dwyer23, Keith K Stanley1 and Jonathan R Iredell23*

Author Affiliations

1 AusDiagnostics Pty Ltd, 3/36 O'Riordan Street, Alexandria, NSW 2015, Australia

2 Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Westmead NSW 2145, Australia

3 Institute for Clinical Pathology and Medical Research, Westmead Hospital Westmead NSW 2145, Australia

4 Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000; Australia

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BMC Infectious Diseases 2010, 10:113  doi:10.1186/1471-2334-10-113

Published: 11 May 2010



Influenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak.


We developed a multiplexed PCR (MT-PCR) assay for the simultaneous diagnosis of respiratory viruses causing influenza-like illness, including the specific recognition of influenza A haemagglutinin subtypes H1, H3, and H5. We tested several hundred clinical specimens in two diagnostic reference laboratories and compared the results with standard techniques.


The sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens. The MT-PCR assays provided differential diagnoses as well as potentially useful quantitation of virus in clinical samples.


MT-PCR is a potentially powerful tool for the differential diagnosis of influenza-like illness in the clinical diagnostic laboratory.