Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate

Abstract

Background: Lactobacillus 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 identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results: To select species-specific sequences or genes, a total of 180 Lactobacillus 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 polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. 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. Conclusions: The 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.

Deciphering Bacterial Community Structure, Functional Prediction and Food Safety Assessment in Fermented Fruits Using Next-Generation 16S rRNA Amplicon Sequencing

“…However, these genera mostly comprised non-pathogenic species. Several studies have demonstrated that the accuracy of PCR assays using appropriate primers for bacterial identification at the species level [ 69 , 70 ]. The PCR analysis revealed that only S. aureus was detected in one sample.…”

The mutL Gene as a Genome-Wide Taxonomic Marker for High Resolution Discrimination of Lactiplantibacillus plantarum and Its Closely Related Taxa

“…However, because several phylogenetically closely related novel L. plantarum group species have been proposed in recent years [ 7 , 8 ], the usability and specificity of recA gene–based PCR and sequencing as well as species-specific primers should be reassessed through in silico methods. To identify highly distinguished taxonomic markers with deep-level phylogeny or species-specific genes according to the gain and loss of variable regions, the use of genome comparison methods have been applied to resolve the phylogenetically and phenotypically closely related species; this serves as an alternative to the conventional universal phylogenetic targets [ 55 , 56 , 57 , 58 , 59 , 60 ].…”

Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers

“…In previous studies, genes such as 16S rRNA, 16S-23S rRNA intergenic spacer region, and the elongation factor Tu (tuf) gene have been used to distinguish microorganisms at the species or subspecies level [4,9,[26][27][28][29]. However, some studies have reported that these genes share high sequence similarities without showing sufficient variabilities to allow for the differentiation between L. delbrueckii subspecies [4,13]. In contrast, we selected genetic markers specific to the genomes of each subspecies using pangenome analysis.…”