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

S. Arthur Sylvester, J. M. Kataria, K. Dhama, N. Senthilkumar and S. Tomar *
Division of Avian Diseases, Indian Veterinary Research Institute Izatnagar - 243 122 (U.P.)
*Avian Medicine Section, Central Avian Research Institute, Izatnagar - 243122 (U.P.)

The present study was carried out to standardize reverse transcriptase - polymerase chain reaction (RT-PCR) for the detection of IBV in infected allantoic fluid using virus specific degenerate primers, amplifying the whole S1 (spike) gene of IBV, for detection of clinical cases of avian infectious bronchitis virus (IBV) infection and its application in the diagnosis of the disease, which is prevalent in our country. Three IBV isolates originating from different clinical cases of IB from Ajmer, Gurgaon and Mathura 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 cDNA generated out of the template RNA by reverse transcription, from the IBV infected allantoic fluid facilitated a 1791bp specific product of the expected size. The RT-PCR technique was optimized for detection of IBV from infected allantoic fluid and consistent results were obtained with the following PCR cyclic conditions- initial denaturation at 95 0C for 5 min, followed by 30 cycles of denaturation at 94 0C for 1 min, primer annealing at 43 0C for 90 sec, extension at 72 0C for 2 min and a final extension at 72 0C for 15 min. 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 degenerate PCR primers detected specifically the IBV RNA only, and when tested with the DNA/RNA of other avian pathogens viz. fowl pox virus , lymphoid leucosis virus, fowl adenovirus serotype 4, egg drop syndrome 1976 virus, chicken anaemia virus, Newcastle disease virus, infectious bursal disease virus, avian reovirus, Escherichia coli, Pasteurella multocida, Mycoplasma gallisepticum (S-6 strain) and Salmonella Gallinarum, yielded negative results. In conclusion, the optimization of a 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.

Source : IPSACON-2005

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