Genome-based species-specific primers for rapid identification of six species of Lactobacillus acidophilus group using multiplex PCR

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.

Lactobacillus crispatus DSM25988 as novel bioactive agent to co-aggregate Streptococcus pyogenes and to exclude it by binding to human cells

“…Lactobacillus crispatus is a commensal gram-positive lactic acid bacterium inhabiting the human vaginal and intestinal mucosa, and several strains are highly adhesive to different types of epithelial cells (He et al, 2020). Phylogenetically, L. crispatus is closely related to Lactobacillus acidophilus and Lactobacillus helveticus, two of the most commonly used probiotics in food industry (You and Kim, 2020). L. crispatus has a long history of human consumption and has been identified in starter cultures for rye sourdough, in cheese, wine, sauerkraut and in whisky (Beganovic et al, 2014;Henri-Dubernet et al, 2008;Kačániová et al, 2012;Meroth et al, 2003;Van Beek and Priest, 2000).…”

PhyloPrimer: a taxon-specific oligonucleotide design platform

“…It is also possible to target non-universal genes, such as the nifH gene (e.g., Gaby & Buckley, 2012 ) and the pmoA gene (e.g., Wang et al, 2017 ), in order to target only organisms with a specific metabolism and that occupy specific environmental niches. Oligos can also have a more specific target: they can amplify only genes present in organisms of interest even when the gene is present in a wider selection of organisms (e.g., You & Kim, 2020 ; Yu et al, 2005 ). When no predesigned oligos are available, however, it is necessary to develop new ones.…”