Abstract
Many Lactobacillus species are frequently isolated from dairy products, animal guts, and the vaginas of healthy women. However, sequencing-based identification of isolated Lactobacillus strain is time/cost-consuming and lobor-intensive. In this study, we developed a multiplex PCR method to distinguish six closely related species in the Lactobacillus acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, and L. gallinarum), which is based on species-specific primer sets. Altogether, 86 genomes of 9 Lactobacillus species from the National Center of Biotechnology Information (NCBI) database were compared to detect species-specific genes and design six species-specific primer sets. The PCR conditions of the individual primer sets were optimized via gradient PCR methods. A final multiplex PCR condition was also optimized for a mixture of all six primer sets mixed. When identifying a single strain, the optimized multiplex PCR method can specifically detect one of the six species, but no band was amplified at least from the other Lactobacillus and Enterococcus species. These results indicated that species-specific primer sets designed from the genome comparison could identify one strain within the six Lactobacillus species by a single PCR reaction. Using the method described here, we will be able to save time, cost, and labor during species identification and screening of commercially important probiotic lactobacilli.
Highlights
Lactic acid bacteria (LAB) are of great economic importance to the food biotechnology sector as well as in the production of other health supplements [1,2,3,4,5]
We investigated species-specific primer sets that were used to identify 6 species of L. acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, L. gallinarum) via comparative genomics
Members of the Lactobacillus acidophilus group demonstrated that their sequences were similar and formed one branch (S1 Fig)
Summary
Lactic acid bacteria (LAB) are of great economic importance to the food biotechnology sector as well as in the production of other health supplements [1,2,3,4,5]. The Lactobacillus acidophilus homology group is a clade of homologous Lactobacillus species categorized using molecular-based phenotypic and genotypic techniques [10,11]. Species-specific primers for identification of six species of Lactobacillus acidophilus group within the same homology group share similar genomic patterns; phenotypes and functions are species dependent [12,13]. There is a growing interest towards bacterial characteristic identification at the species and strain level. There are several methods to identify bacteria via phenotypes and biochemical characteristics, they are often ambiguous and steeped in uncertainty
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