Open Access Highly Accessed Research article

Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

Lisa FP Ng1, Ian Barr2, Tung Nguyen3, Suriani Mohd Noor4, Rosemary Sok-Pin Tan5, Lora V Agathe1, Sanjay Gupta1, Hassuzana Khalil4, Thanh Long To3, Sharifah Syed Hassan4 and Ee-Chee Ren16*

Author Affiliations

1 Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, 138672, Singapore

2 WHO Influenza Centre, 45 Poplar Road, Parkville, Melbourne, 3052, Australia

3 Virology Section, National Centre for Veterinary Diagnostics, Department of Animal Health, Vietnam

4 Virology Section, Veterinary Research Institute, 31400, Ipoh, Perak, Malaysia

5 Veredus Laboratories Pte Ltd, 83 Science Park Drive, #03-02A The Curie, Singapore Science Park 1, 118259, Singapore

6 Department of Microbiology, Faculty of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, 117597, Singapore

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BMC Infectious Diseases 2006, 6:40  doi:10.1186/1471-2334-6-40

Published: 2 March 2006



Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus.


A one-step reverse-transcription PCR assay was developed to detect the H5N1 avian influenza A virus. The specificity of the assay was shown by testing sub-types of influenza A virus and other viral and bacterial pathogens; and on field samples.


Validation on 145 field specimens from Vietnam and Malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. Detection was 100% from allantoic fluid in H5N1 positive samples, suggesting it to be a reliable sampling source for accurate detection.


The assay developed from this study indicates that the primers are specific for the H5N1 influenza virus. As shown by the field tested results, this assay would be highly useful as a diagnostic tool to help identify and control influenza epidemics.