Development and application of a SNP-based strain-specific identification PCR method for Bifidobacterium animalis...

Accurate strain-specific identification of probiotics is critical for ensuring product efficacy and safety. However, many established techniques, including whole-genome sequencing, pose challenges for industrial application due to their cost, speed, and practicality limitations. To resolve this gap, we developed a novel strain-specific PCR coupled with Sanger sequencing method for Bifidobacterium animalis subsp. lactis BL-99 by integrating pan-genomic SNP screening with optimized primer design. Our approach identified a unique guanine SNP that distinguishes BL-99 from other B. animalis strains. Validation demonstrated 100% specificity across 55 microbial strains, a detection limit of 10⁵ CFU/mL in pure cultures, and consistent performance in industrial fermentation samples. This method reduces identification costs and prevents mislabeling risks in commercial probiotics, providing dairy manufacturers with a rapid tool for quality control and regulatory compliance.