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

作者：Kuriakose Teneema; Hilt Deborah A; Jackwood Mark W^*

来源：Avian Diseases, 2012, 56(1): 90-96.

DOI：10.1637/9828-060211-Reg.1

• avian influenza virus
• diagnosis
• multiplex microsphere assay
• subtyping

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 X 10(8) to 1 X 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 X 10(2.5) to 1 X 10(3.2) 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, NI, and N2 subtypes.

• 出版日期2012-3