Validation of probiotic species or subspecies identity in commercial probiotic products using high-resolution PCR method based on large-scale genomic analysis

• This is the first study to develop a high-resolution PCR method for 25 species. • Pangenome analysis revealed molecular marker genes specific to each species. • The primer pairs showed 100% specificity and sensitivity in real-time PCR. • Some commercial products observed the absence of declared species on the label. • This assay was successfully applied to monitor commercial probiotic products. The health-promoting effects of probiotics are species-specific, and hence it is important to declare the correct information in products. However, some studies have identified issues related to the accuracy of labeling commercial probiotic products. In this study, we developed a high-resolution real-time PCR method based on pangenome analysis for a more affordable, rapid, and accurate identification of commercial probiotic products than sequencing methods. We selected 25 species or subspecies primarily used for probiotic strains and are closely related to them as targets. To extract molecular markers, 354 whole-genome sequences present in the target genomes but not in the pangenome of other genomes were compared, which resulted in the identification of molecular marker genes. The marker genes exhibited 100% specificity for 100 strains as assessed by the real-time PCR method. Fifty probiotic and dairy products were investigated to verify the information claimed on the label. Real-time PCR results showed that most products reflected the bacterial species declared in the label claim, whereas 12 products showed the presence of undeclared species or missing species. Our method for accurately verifying the labeling of probiotic products would be useful for quality control and safety.