Development and multi-platform validation of a pan-genomic-driven PMA-qPCR method for the precise quantification of...

Accurate quantification of viable Bifidobacterium animalis subsp. lactis is essential for probiotic quality and functionality, yet remains challenging in complex multi-strain products and diverse matrices. Here, we developed a pan-genomic-driven propidium monoazide-quantitative PCR (PMA-qPCR) assay, enabling robust and precise enumeration of viable B. lactis in both single-strain and complex formulations. Using a pan-genomic approach combined with two-tiered in silico and experimental validation, we identified a novel subspecies-specific target, the pyridoxal phosphate homeostasis protein, which allowed exclusive detection without cross-reactivity among 36 non-target strains, including closely related taxa. The optimized PMA protocol demonstrated high strain-independent universality, effectively excluding signals from non-viable cells while maintaining sensitivity (LOD 10³ CFU/mL) and strong linearity (R² > 0.99, 10³-10⁸ CFU/mL) across diverse sample matrices and excipients. Importantly, the method enabled accurate quantification of B. lactis in complex multi-strain probiotic products, and in long-stored samples, PMA-qPCR detected higher viable counts than traditional plate counting; this was corroborated by flow cytometry and single-cell D₂O-labeled Raman spectroscopy, revealing physiologically intact cells beyond culturability. Collectively, these findings establish the pan-genomic-driven PMA-qPCR as a culture-independent, high-resolution tool for reliable quality control and label verification of B. lactis, supporting standardized testing, regulatory compliance, and multidimensional viability assessment in increasingly complex commercial probiotic products.