Detection and identification of Lactobacillus acidophilus species and its commercial probiotic strains using CRISPR loci-based amplicon analysis

Abstract

The market for probiotics has grown exponentially in recent decades. Owing to the diversification of probiotic products, researchers who focus on issues related to their quality, safety, and efficacy have highlighted the need to identify specific probiotic strains, such as Lactobacillus acidophilus. However, phylogenetic analyses based on 16S rRNA gene sequence differences cannot distinguish L. acidophilus strains or some Lactobacillus species. Recent studies have demonstrated the value of clustered regularly interspaced short palindromic repeats (CRISPR)-based methodologies for bacterial genotyping. Here, we developed CRISPR loci-based L. acidophilus species- and strain-specific markers. The L.acidophilus species-specific primer set produced a 267-bp PCR product in three Lactobacillus species (L. gallinarum, L. helveticus, and L. amylovorus). The L.acidophilus strain-specific primer set produced 877-, 938-, and 205-bp products in L. acidophilus ACTT 4356T, NCFM, and LA1_11869BP, respectively. The PCR limit of detection was 0.1–0.01 ng of genomic DNA. Furthermore, this study confirmed that the primer sets could amplify L. acidophilus species and subgroup strains in commercial yogurt products. These novel markers can be used to easily and accurately identify L. acidophilus in the food and dairy industries.