Establishment of RT-PCR for detection of avian influenza virus (H9N2) in field cases compared to virus isolation method

Avian influenza (ai) is one of the most economical viral diseases in iranian poultry industry because of increased mortality and declines in egg production in affected chicken flocks. virus isolation in embryonated spf chicken eggs, a time consuming method, is the most common diagnostic test. in this study, virus isolation and rt-pcr for detection of avian influenza virus were evaluated by use of trachea samples collected from ten clinically affected broiler flocks. ten percent trachea tissue suspension was inoculated to 10-day-old embryonated spf chicken eggs. avian influenza virus, h9n2 subtype, was confirmed in dead embryos by hemagglutination inhibition test. simultaneously, total rna was extracted from the trachea tissues and rt-pcr was optimized for amplification of a 488 bp dna fragment of h9n2 subtype hemagglutinin gene. in seven of the ten flocks both the rt- pcr and virus isolation methods detected the virus. in two flocks ai virus was not detected by either method, while in a flock ai virus was identified only by rt-pcr. newcastle disease virus was isolated in one of the flocks in which was negative by both the methods. the sensitivity and specificity rate by rt-pcr in comparison with virus isolation as gold standard test was coordinately 100% and 66%, respectively. the detection of ai by rt- pcr showed the best correlation with the virus isolation method even more sensitive than it. it is suggested that in surveillance centers where early detection of ai virus is goal, rt-pcr is a reliable method for rapid laboratory diagnosis of influenza in dead chickens because of its speed, sensitivity and specificity.