Development and validation of a propidium monoazide-qPCR assay with matrix-matched calibration for selective quantification of viable Limosilactobacillus reuteri in multi-strain probiotic powders

Accurate enumeration of viable probiotic cells in commercial products is essential for quality control, label compliance, and product development. Conventional plate counting is labour-intensive and primarily quantifies culturable cells, whereas quantitative PCR (qPCR) is rapid, strain-specific, and detects DNA from both live and dead cells. We developed and validated a strain-specific propidium monoazide (PMA)-qPCR assay targeting a unique genomic region of Limosilactobacillus reuteri IDCC 3701 (RE), an industrial probiotic strain used in multi-strain products. The assay was evaluated in pure and mixed cultures and in single- and four-strain freeze-dried probiotic powders, using calibration curves generated directly from the RE freeze-dried powder to reduce matrix mismatch. PMAxx dye treatment effectively suppressed amplification of dead-cell DNA (99.985% inhibition) with minimal impact on viable-cell amplification. The assay enabled strain-specific quantification of viable RE in cultures from 10 4 –10 9 CFU/mL, and powder-based quantification was performed within a validated linear range of ≥10 5 CFU/mL. This approach provides a practical tool for RE-containing probiotic quality control and a useful framework for extending powder-calibrated PMA-qPCR to other industrial strains.