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1 March 2012 Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay
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Abstract

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase–PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 × 108 to 1 × 109 copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 × 102.5 to 1 × 103.2 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

American Association of Avian Pathologists
Teneema Kuriakose, Deborah A. Hilt, and Mark W. Jackwood "Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay," Avian Diseases 56(1), 90-96, (1 March 2012). https://doi.org/10.1637/9828-060211-Reg.1
Received: 2 June 2011; Accepted: 1 September 2011; Published: 1 March 2012
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