Abstract
BackgroundLactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. ResultsTo select species-specific sequences or genes, a total of 143 Lactobacillus complete genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. ConclusionsThe method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.
Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis