Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis.

Latilactobacillus sakei group comprises four closely related species, making it difficult to accurately distinguish them with standard markers such as the 16S rRNA gene. The objective of our study was to mine novel markers for PCR detection and discrimination of L. sakei group species and L. sakei subspecies by comparative pan-genomic analysis. A total of 63 genome sequences of L. sakei group species consisted of 119,899 coding genes, yielding 5741 pan-genomes, 831 core-genomes, 3347 accessory-genomes, and 1563 unique-genomes. The accessory-genome was compared to extract unique candidate genes common only to genomes of the same species. The candidate genes were then aligned with the other bacterial genomes to select marker genes present in all genomes of a given species, but not in the genomes of other species. We identified the arginine/ornithine antiporter, putative cell surface protein precursor, sodium:solute symporter, PRD domain protein, PTS sugar transporter subunit IIC, and phosphoenolpyruvate-dependent sugar phosphotransferase system EIIC as marker genes for L. sakei, L. sakei subsp. sakei, L. sakei subsp. carnosus, L. curvatus, L. graminis, and L. fuchuensis, respectively. Primer pairs were designed for each marker and showed 100% specificity for 48 lactic acid bacterial reference strains. The PCR method developed in this study was used to evaluate 106 strains isolated from fermented foods to demonstrate that the marker genes provided a viable alternative to the 16S rRNA gene. We also applied the method to the monitoring of kimchi samples to quantify L. sakei group species or subspecies. Our PCR method based on novel markers can rapidly identify L. sakei group with high accuracy and high throughput.