Comparative Analysis of PCR Primers Used for Detection of Infectious Bronchitis Virus (IBV) Variant ۲ in Iran and Neighboring Countries: Evaluation of Their Specificity for Reference Strains

Background: Infectious bronchitis virus (IBV) remains one of the most economically important pathogens in poultry, with numerous genotypes circulating worldwide. Among them, the Variant ۲ (GI-۲۳) lineage originally identified in the Middle East has shown high genetic and antigenic diversity, raising questions about diagnostic accuracy in regional studies. The present work aimed to evaluate whether PCR primers used in Iran and neighboring countries are specific to established prototype strains, or whether primer mismatch could lead to misidentification of Variant ۲ isolates or other mutants. Methods: Eighteen primer pairs (labeled A to R) were analyzed by both in silico nucleotide alignment and NCBI Primer-BLAST, using partial S۱ gene or complete genome sequences of prototype strains: Egypt/۱۲۱۲B/۲۰۱۲ (EG/۱۲۱۲B), Israel/۱۴۹۴/۰۶ (IS/۱۴۹۴/۰۶), Massachusetts ۴۱ (Mass ۴۱), ۷۹۳/B, and QX (GI-۱۹). Alignment analyses were performed to assess nucleotide complementarity, and Primer-BLAST was used to confirm potential amplification targets. Results: The results indicated that only a limited number of primer pairs demonstrated specific and consistent amplification with reference strains. Primer A showed positive alignment and Primer-BLAST results with the QX strain. Primers C, J, K, D, and Q produced positive results with the Israel and ۷۹۳/B reference strains, while Primers F and G matched the Massachusetts strain. Several primers showed no significant binding or amplification across all reference strains, although minor nonsignificant similarities were noted in nucleotide alignment. Conclusion: These findings highlight that some primers used regionally may not truly represent variant-specific diagnostic tools. The study emphasizes the necessity of primer validation against reference genomes before diagnostic application to avoid overestimation of Variant ۲ circulation in the region.

کلیدواژه ها:

Infectious bronchitis virus ، Variant ۲ ، PCR primers ، S۱ gene ، Iran ، Middle East