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

Abstract

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.

Keywords: VP5 gene; alignment of sequences; infectious bursal disease virus; novel reverse transcriptase polymerase chain reaction; virulent strains.

Figure-1

Clustal omega alignment results of the IBDV-Vp5 sequences available on GenBank (https://www.ncbi.nlm.nih.gov/genbank/). A more than 405-bp coincidence with most of the other sequences was observed (accession number AF165151.1). IBDV=Infectious bursal disease virus.

Figure-2

Results of the primer annealing test. A 402 bp band is consistently visible in all evaluated temperatures, but the most visible band is present in the lane of the temperature of 61°C.

Figure-3

Evaluation of limit of detection test. Our newly designed primers can successfully detect bursal infectious disease virus RNA at concentrations of 0.125 M.

Figure-4

Results of the primer concentration test. Our newly designed primers can successfully detect bursal infectious disease virus RNA at a concentration of 0.125 µM.

Figure-5

Results of specificity test. Lane 1=O’GeneRuler DNA ladder mix, Lane 2=Empty, Lane 3=Pure CEF total RNA, Lane 4=Infectious bronchitis virus positive sample, Lane 5=Infectious laryngotracheitis virus positive sample, Lane 6=Avian metapneumovirus positive sample, Lane 7=Newcastle disease virus positive sample, Lane 8=Positive sample of avian adenovirus responsible for inclusion body hepatitis. Lane 9=Chicken anemia virus-positive sample, Lane 10=Avibacterium paragallinarum-positive sample, Lane 11=PCR grade water, Lane 12=Infectious bursal disease virus-positive sample, PCR=Polymerase chain reaction.