Development of a csp-based RT-qPCR method for specific quantification of Bifidobacterium animalis by comparative...

The current standard for quantitative detection of probiotics by plate counting method (PCM) has significant drawbacks, including high variability and the inability to differentiate specific strain types in mixed microorganisms. In this work, a real-time quantitative reverse transcription PCR (RT-qPCR) method was developed to quantitatively detect Bifidobacterium animalis. Comparative genomic analysis revealed significant differences in collinearity among various Bifidobacterium species and identified five locally co-linear blocks (LCBs) potentially containing unique species-specific genes. Among five pairs of primers designed targeting these low-collinearity genomic regions, only the csp (cell surface protein) primer pair showed good specificity for B. animalis and not for other species. The csp-based RT-qPCR method (csp method) successfully detected B. animalis, with a low limit of detection (LOD) of 7.2 × 10¹ CFU/mL and a relative standard deviation (RSD) of 1.08 % compared to PCM. We conducted applicability tests of the csp method in dairy products containing multiple strains spiked with the same amount of B. animalis. Viable counts of B. animalis determined by the csp method showed no significant difference between skimmed milk and fermented milk samples, while viable counts obtained by PCM and the reported recA method were significantly higher than those from the csp method. Therefore, our developed csp method shows potential as an alternative to PCM for accurate detection of B. animalis in complex food matrices containing various microorganisms.