The Single-Step Multiplex Reverse Transcription- Polymerase Chain Reaction Assay for Detecting H5 and H7 Avian Influenza A Viruses

Abstract

Avian influenza (AI) A virus subtypes H5 and H7 cause severe disease in domestic poultry, including chickens and turkeys. Moreover, H5 and H7 AI A viruses can cross the species barrier from poultry to humans. In the present study, we have developed a single-step multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) for detecting H5 and H7 AI A viruses. This assay was applied to the poultry isolates with the aim of establishing a surveillance method to monitor possible transmission to humans. Two subtype-specific primer sets capable of producing PCR products of 157 and 326 base pairs corresponding to AI A virus H5 and H7 subtypes, respectively, were utilized in a one-step and one-tube reaction. The single-step multiplex RT-PCR assay developed in this study was found to be specific for detecting H5 and H7 AI A viruses. No specific amplification bands were detected with total nucleic acids extracted from other influenza hemagglutinin subtypes and other viral pathogens. The sensitivity of this assay was about 10³ RNA copies/μl. In conclusion, this novel single-step multiplex RT-PCR is a simple assay with high potential for rapid, specific and cost effective laboratory diagnosis of H5 and H7 AI A virus isolates from clinical specimens of poultry.