Identification of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis

A rapid and reliable technique for identifying the strains of lactic acid bacteria (LAB) was developed in this study, and the 16S ribosomal RNA gene (16S rRNA gene) was used as the target. Several species-specific primer pairs were designed based on the variability of 16S rRNA sequence(s) for differentiating 5 strains of lactobacilli which were added into probiotic products in Taiwan. It was simple to identify Lactobacillus acidophilus and L. delbrueckii by species-specific primers, but it could not be used to distinguish L. casei, L. paracasei and L. rhamnosus. Another PCR approach was developed with hybridization probes which were designed according to the difference among the 16S rRNA genes of L. casei, L. paracasei and L. rhamnosus, and melting curve analysis of the hybridization probe was used to distinguish them. It was found that this approach could identify L. paracasei and L. rhamnosus correctly but not separate L. paracasei from L. casei, the result was due to both of them had the same 16S rRNA sequence. These results suggest that melting curve analysis of PCR approach in this study is a rapid, simple and accurate method in distinguishing the closely related strains of lactobacilli.