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Journal of Clinical Microbiology, January 2009, p. 86-92, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01090-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR {triangledown}

Wei Wang,1 Peijun Ren,1 Sek Mardi,2 Lili Hou,1 Cheguo Tsai,1 Kwok Hung Chan,3 Peter Cheng,4 Jun Sheng,5 Philippe Buchy,2 Bing Sun,1 Tetsuya Toyoda,1 Wilina Lim,4 J. S. Malik Peiris,3,6 Paul Zhou,1 and Vincent Deubel1*

Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Shanghai, China,1 Institut Pasteur du Cambodge, Phnom Penh, Cambodia,2 Department of Microbiology, the University of Hong Kong, Hong Kong,3 Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong,4 Shanghai Nanxiang Hospital, Shanghai, China,5 HKU-Pasteur Research Centre, Hong Kong SAR6

Received 9 June 2008/ Returned for modification 13 August 2008/ Accepted 13 October 2008

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.

* Corresponding author. Mailing address: Unit of Emerging Viruses, Chinese Academy of Sciences, Institut Pasteur of Shanghai, 411 Hefei Road, 200025 Shanghai, People's Republic of China. Phone: 86 21 6384 5146. Fax: 86 21 6384 3571. E-mail: vdeubel{at}sibs.ac.cn

{triangledown} Published ahead of print on 29 October 2008.

Journal of Clinical Microbiology, January 2009, p. 86-92, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01090-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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