Development of RT-PCR Using External and Internal Positive Controls Based on 5 Untranslated Region (UTR) for Molecular Detection of Avian Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) belongs to the group of gamma coronaviruses along with other avian coronaviruses. The disease caused by IBV can appear similar to infectious laryngotracheitis, avian influenza, and velogenic Newcastle disease, which are high priority diseases. The clinical signs can be accompanied by mortalities in broiler chickens and reduced eggshell and albumin quality in layer hens, leading to economic loss for the poultry industry. Rapid detection of IBV is useful for implementation of control measures, research purposes, and understanding the epidemiology and evolution of IBVs. The aim of the present study was the rapid identification of IB with the molecular method, which targets the 5’ untranslated region (UTR) gene of IBV that is less variable than the other genes, with homologies greater than 90% among IBV strains.
The primers designed to amplify a conserved fragment of the gene. Analytical sensitivity and specificity of the assay were determined.
The results of specificity exhibited the specific amplification of the designed primers for IBV. Sensitivity was 10 pg/μl of the pTZ57R/T-5' UTR. This is the first report of RT-PCR method coupled with construction of comparative internal positive control (IPC) according to 5´UTR gene for accurate detection of IBV. 100 fg/μl of the IPC amplified in the presence of the limit of detection (10 pg/μl) of 5' UTR gene was determined as the optimal concentration of IPC plasmid for RT-PCR of clinical specimens.
The RT-PCR assay presented provides a time saving, sensitive, and reliable method for detection of IBV.