Standardization and application of reverse transcriptase - polymerase chain reaction (RT-PCR) for detection of avian infectious bronchitis virus

The present study was carried out to standardize reverse transcriptase - polymerase chain reaction (RT-PCR) for the detection of avian infectious bronchitis virus (IBV) in infected allantoic fluid using virus specific degenerate primers, amplifying the whole SI (spike) gene of IBV, for detection of clinical cases of IBV infection, and its application in the diagnosis of the disease, which is prevalent in our country. Six field IBV isolates originating from clinical cases of IB from various geographical locations were passaged in specific pathogen free (SPF) embryos for the standardization of RT-PCR protocol to obtain confirmatory diagnosis of IBV. All the isolates showed characteristic lesions of IBV viz. curling and dwarfing of the embryos. Successful RT-PCR amplification, using eDNA generated out of the template RNA by reverse transcription, from the IBV infected allantoic fluid yielded a 1600 bp specific product of the expected size. Detection limit of the developed RT-PCR technique for the IBV RNA was found to be 1 pg indicating the test to be highly sensitive. The optimized rapid and reliable technique of RT-PCR could prove to be a highly sensitive and specific detection system for diagnosing IBV field infections in chickens.