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Development and Validation of a One-Step Real-Time PCR Assay for Simultaneous Detection of Subtype H5, H7, and H9 Avian Influenza Viruses

Istituto Zooprofilattico Sperimentale delle Venezie, Research and Development Department, OIE/FAO and National Reference Laboratory for Newcastle Disease and Avian Influenza, OIE Collaborating Centre for Epidemiology, Training, and Control of Emerging Avian Diseases, Viale dell'Università 10, Legnaro, Padova, Italy

Received 14 November 2007/ Returned for modification 9 January 2008/ Accepted 17 March 2008

Among the different hemagglutinin (HA) subtypes of avian influenza (AI) viruses, H5, H7, and H9 are of major interest because of the serious consequences for the poultry industry and the increasing frequency of direct transmission of these viruses to humans. The availability of new tools to rapidly detect and subtype the influenza viruses can enable the immediate application of measures to prevent the widespread transmission of the infection. In this study, a novel one-step real-time reverse transcription-PCR (RRT-PCR) was developed to detect simultaneously the H5, H7, and H9 subtypes of AI viruses from clinical samples of avian origin. The sensitivity of the RRT-PCR assay was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection ranging from 10¹ to 10³ RNA copies and from 10¹ 50% egg infectious dose (EID₅₀)/100 µl to 10^2.74 EID₅₀/100 µl with titrated viruses. Excellent results were achieved in the intra- and interassay variability tests. The comparison of the results with those obtained from the analysis of 725 avian samples by means of the reference method (virus isolation [VI]) showed a high level of agreement. To date, this is the first real-time PCR protocol available for the simultaneous detection of AI viruses belonging to subtypes H5, H7, and H9, and the results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of the three most-important AI virus subtypes in samples of avian origin.

Published ahead of print on 26 March 2008.