Development of a Novel Multiplex Multiple Oligonucleotide Ligation PCR System for Identification of Probiotic Bacteria in Food Supplements

Probiotic food supplements are widely consumed, yet their quality and labeling accuracy remain inconsistent due to variable manufacturing and insufficient regulation. Conventional methods, e.g., culture and qPCR, enable detection of only a few microorganisms per assay, restricting comprehensive analysis. To overcome this, we developed a multiplex multiple oligonucleotide ligation PCR (MOL-PCR) assay for simultaneous detection of 29 bacterial and yeast species found in probiotic supplements. Protocol improvements─preamplification, optimized moligo concentrations, and a novel ligation product purification─enhanced sensitivity and reduced background noise. The assay achieved 100% analytical specificity with a detection limit of ∼10¹-10² microorganisms per reaction. Validation on 35 commercial supplements showed strong concordance with qPCR, with a diagnostic sensitivity of 96.4% and a specificity of 98.1%. Importantly, the method revealed frequent discrepancies between declared and actual product composition, including missing strains and undeclared contaminants. This high-throughput assay provides a robust tool for probiotic quality control and regulatory compliance.