Matrix-matched standard curves reduce overestimation in PMAxx-qPCR viable counting of Lacticaseibacillus rhamnosus in multi-strain probiotic powders

Strain-level enumeration of viable cells is critical for the quality control of multi-strain probiotic products. However, culture-based colony-forming unit (CFU) counting is time-consuming and often lacks sufficient discriminatory power among closely related species and strains. In this study, we developed a strain-specific viability quantitative PCR assay by coupling PMAxx treatment with qPCR (PMAxx-qPCR) to selectively quantify Lacticaseibacillus rhamnosus IDCC 3201 (RH) in single-strain and mixed probiotic preparations. RH-specific primers designed using comparative genomics produced a single melt-curve peak for RH and showed no detectable cross-reactivity with the three non-target probiotic strains. PMAxx effectively suppressed amplification of DNA derived from heat-inactivated RH cells, achieving 99.81% inhibition while minimally affecting the signals from live RH. PMAxx-qPCR enabled linear quantification of RH at concentrations ≥ 3.78 log CFU/mL in both single and mixed cultures. The Ct values were consistently elevated in mixed cultures, suggesting matrix-related interference under mixed-strain conditions. In freeze-dried powders, matrix-matched calibration substantially improved the agreement with plate counts compared to a broth-based standard curve. The mean bias was reduced from 1.54 to 0.25 log CFU/mL. In multi-strain freeze-dried powders, PMAxx-qPCR modestly underestimated RH counts relative to formulation-based theoretical values, suggesting residual interference in the complex matrices. Overall, this PMAxx-qPCR assay provides a rapid, viability-oriented, strain-resolved approach for RH enumeration and underscores the importance of matrix-matched standardization for accurate molecular quantification in probiotic powders.