Typing infectious bronchitis virus strains using reverse transcription-polymerase chain reaction and restriction fragment length polymorphism analysis to compare the 3′ 7.5 kb of their genomes

Abstract

Typing infectious bronchitis virus (IBV) strains is useful for implementation of control measures and for understanding the epidemiology and evolution of IBV. The aim of the work reported here was to develop a rapid and sensitive method for typing isolates of IBV, if possible directly from tissues of infected birds. A procedure was developed for differentiation of IBV strains by restriction endonuclease fragment length polymorphism (RFLP) analysis of a 7.5 kb DNA fragment amplified from their genome by reverse transcription-polymerase chain reaction (RT-PCR). This fragment encompassed all of the genes encoding the structural proteins of the virus. Viral RNA was extracted either directly from tissues of diseased birds or from virus propagated in embryonated eggs, and was subjected to RT-PCR. Three different restriction endonucleases, AluI, Sau3AI and MnlI, were used to digest the 7.5 kb PCR product from different IBV strains and the resultant RFLP patterns were compared. Patterns obtained with all three enzymes grouped IBV strains belonging to the same serotype in the same cluster. These results show that the RT-PCR-RFLP system described here can be used as a quick and inexpensive tool for differentiating IBV strains.