| Summary: | Viability is a prerequisite for any therapeutic benefits associated with the ingestion of probiotic bacteria. Current culture-based techniques are inadequate for the enumeration of probiotics in mixed-species food products. This study utilized a quantitative PCR (qPCR) method coupled with propidium monoazide (PMAxx), and novel species-specific tuf gene primers to selectively enumerate Lacticaseibacillus rhamnosus, Bifidobacterium spp., and yogurt starter cultures in mixed-species probiotic yogurt. The method was optimized for PMAxx concentration and specificity and evaluated for efficiency and applicability. PMAxx-qPCR showed high specificity to the target organisms in mixed-species yogurt, quantifying only viable cells. The linear dynamic ranges were established over five to seven orders of magnitude. The assay was reliable with an efficiency of 91–99%, R2 values > 0.99, and a good correlation to the plate count method (r = 0.882). The results of this study demonstrate the high selectivity, improved lead time, and reliability of PMAxx-qPCR over the culture-dependent method, making it a valuable tool for inline viability verification during processing and improving probiotic quality assurance for processors and consumers.
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