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Journal of Clinical Microbiology, November 2001, p. 4097-4102, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.4097-4102.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Combined PCR-Heteroduplex Mobility Assay for Detection and Differentiation of Influenza A Viruses from Different Animal Species

Joanna S. Ellis* and Maria C. Zambon

Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, Colindale, London NW9 5HT, United Kingdom

Received 19 December 2000/Returned for modification 13 January 2001/Accepted 8 September 2001

Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The early detection and identification of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from different animal species, a combined reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sensitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were differentiated by HMA. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It was found to be most closely related to the avian A/Quail/HongKong/G1/97 H9N2 reference PCR product. Sequence analysis showed a nucleotide divergence of 1.1% between the A/Quail/HongKong/G1/97 and A/HongKong/1073/99 amplicons. From the results of our work, we consider the RT-PCR HMA method described to offer a rapid and sensitive means for screening for novel or unusual influenza viruses.

* Corresponding author. Mailing address: Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Lab., Public Health Lab. Service, Central Public Health Laboratory, 61 Colindale Ave., Colindale, London NW9 5HT, United Kingdom. Phone: 020 8200 4400. Fax: 020 8200 1569. E-mail: jellis{at}phls.nhs.uk.

Journal of Clinical Microbiology, November 2001, p. 4097-4102, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.4097-4102.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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