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Rapid Detection of Avian Influenza Virus A and Subtype H5N1 by Single Step Multiplex Reverse Transcription-polymerase Chain Reaction

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Abstract

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

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Acknowledgements

The study was supported by the National Natural Scientific Foundation (30471282) and National Basic Research Program (973)(2005CB523003) and Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan.

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Authors and Affiliations

  1. Department of Infectious Disease, College of Veterinary Medicine, China Agricultural University, 100094, Beijing, People’s Republic of China

    Hui-Ling Wei, Ying-Chun Zhou, Yan-Long Cong, Juan Pu, Shuai Wang & Jin-Hua Liu

  2. Laboratory of Microbiology, Department of Disease Control, Hokkaido University Graduate School of Veterinary Medicine, 060-0818, Sapporo, Japan

    Gui-Rong Bai, Aaron S. Mweene & Hiroshi Kida

Authors
  1. Hui-Ling Wei
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  2. Gui-Rong Bai
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  9. Jin-Hua Liu
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Correspondence to Jin-Hua Liu.

Additional information

The G.R.Bai A.S.Mweene contributed equally to this work

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Wei, HL., Bai, GR., Mweene, A.S. et al. Rapid Detection of Avian Influenza Virus A and Subtype H5N1 by Single Step Multiplex Reverse Transcription-polymerase Chain Reaction. Virus Genes 32, 261–267 (2006). https://doi.org/10.1007/s11262-005-6910-4

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