Development of ddPCR assays for the taxonomic and functional characterization of Lactobacillus acidophilus LA5® in commercial probiotic formulations

Objective: The study focuses on developing rapid and highly sensitive quantitative PCR-based molecular methods for quality control and accurate labeling of commercial probiotic products. Specifically, evaluation of correct taxonomic identification and screening of key functional genes are investigated in different probiotic formulations containing Lactobacillus acidophilus LA5®. Methods: A single-strain lyophilized granule supplement (Brand 1), a single-strain lyophilized capsule (Brand 2), a multi-strain fermented yogurt (Brand 3), and a multi-strain probiotic supplement (Brand 4) were analyzed, starting from DNA extraction using bead-beating technology, followed by purification with silica membrane columns. High quality DNA was used for Droplet Digital PCR (ddPCR) assays, in singleplex and duplex mode with Evagreen, using a primer pair for the taxonomic identification targeting the 16S–23S region and five newly-designed primer pairs targeting functional genes: class A sortase (srtA), choloylglycine hydrolase (bsh1, bsh2), pore-forming S-layer protein (slpA), and class III bacteriocin (LA_01533). Results: The developed ddPCR assays enabled precise quantification of Lactobacillus acidophilus LA5® at both taxonomic and functional levels, for all the investigated probiotic formulations. In singleplex, 16S-23S region and bsh2 genes were quantified with values ranging from 8.30±0.05 to 11.32±0.08 copies/g, and from 8.55±0.08 to 11.11±0.04 copies/g. Duplex ddPCR targeting the additional functional genes yielded values from 8.74±0.01 to 11.09±0.08 (bsh1/LA_01533), and from 8.77±0.03 to 11.23 ± 0.01 (slpA/srtA) copies/g. Conclusions: All six genetic targets were successfully detected in all the probiotic formulations, confirming the method’s robustness for high-sensitivity strain identification, molecular traceability, and consistency with label-claimed functional attributes.