Standardization and validation of a novel reverse transcriptase polymerase chain reaction method for detecting virulent strains of the infectious bursal disease virus

Background and Aim: Gumboro disease is an economically crucial veterinary condition in chickens. It is caused by the infectious bursal disease virus (IBDV). This virus consists of two serotype groups, of which serotype I strain is pathogenic to chickens. For many years, the development of molecular techniques for either diagnostic purposes or surveillance of the appearance of new pathogenic strains has mainly focused on targeting the VP2 genomic region. However, due to the constant necessity for the discrimination between already prevalent vaccine strains and new pathogenic strains of this virus, it becomes imperative to have an immediate molecular method targeting a consensus sequence to achieve this task using field samples to reduce costs. Consequently, we focused on developing a novel reverse transcriptase polymerase chain reaction (RT-PCR) procedure solely for this purpose. Materials and Methods: Eight VP5 sequences were aligned, and the sequence with the majority of nucleotide coincidences was used to design a set of consensus primers. Then, a pathogenic strain of IBDV was propagated in embryonated chicken eggs, and the viral RNA was extracted. Finally, the conditions for this novel RT-PCR were evaluated using a commercial kit and the newly designed primers. Results: After determining the optimal RT-PCR conditions, the newly designed primers successfully amplified a 402-bp consensus sequence of the VP5 gene. In addition, these primers specifically amplified the VP5 sequence of the IBDV-positive samples, not the other samples previously confirmed to be positive for other common poultry pathogens. Conclusion: Our novel RT-PCR procedure has been demonstrated to be helpful in selectively amplifying the consensus sequence of the VP5 gene, indicating that this novel RT-PCR procedure constitutes an important and useful tool to execute initial discrimination of field-retrieved samples containing and not containing virulent strains of this virus before deciding to execute a blindly and more costly sequencing procedure of all the samples together.