Development of a real-time RT-PCR assay specific for the Winterfield 2512 strain of infectious bursal disease virus

Abstract

Cevac® Transmune® IBD was developed by CEVA Santé Animale for vaccination of poultry against infectious bursal disease (IBD, or Gumboro). The vaccine contains the Winterfield 2512 (W2512) strain of infectious bursal disease virus (IBDV). There is currently no rapid method to detect this strain specifically in fowl vaccinated with the Transmune® vaccine. This study describes the development and optimisation of a real-time RT-PCR assay that is sensitive and specific for the W2512 strain in Cevac® Transmune® IBD vaccine. IBDV sequences available publically on Genbank were downloaded and aligned. No sequence unique to IBDV W2512 Segment B was identified in the downloaded sequences, so sequencing of the IBDV W2512 Segment A was undertaken. A single nucleotide polymorphism (SNP) at nucleotide position 2451 of segment A of IBDV W2512 was identified. An assay targeting this SNP was designed and made use of a TaqMan™ minor groove binding (MGB) probe. The optimum primer and probe concentrations of the assay were 200 nM and 100 nM respectively. The efficiency of the assay was 87%, with an R2 of 0.9924. The 95% limit of detection (LOD) was determined through probit analysis (SPSS) of 25 replicates of a two-fold dilution series and determined to be of 4.7x10-5 chicken infection dose (CID50) (95% confidence interval of 1.7x10-5 to 3.4x10-5). The assay was found to be potentially specific after testing other IBDV strains (IBD#6 vero, isolate 1327108, isolate IBDV). However, there is a need to investigate the specificity further by validating the assay with more IBDV field and vaccine strains. The assay was also tested on 12 W2512 positive and 12 W2512 negative impression smears of bursa samples stored on Whatman® FTA cards. All the 12-W2512 negative samples tested negative while 11 out of 12-W2512 positive samples tested positive with this assay. Based on these performance characteristics and findings, the assay is sensitive and could be specific for Cevac® Transmune® IBD’s W2512 vaccine strain. It is rapid, efficient, easy to perform and may be suitable for its intended use, which is to detect W2512 IBDV strain in poultry flock vaccinated with Cevac® Transmune® IBD vaccine.