Bacterial strains and probiotic and dairy products
The Lactobacillus reference strains were obtained from the Korean Collection for Type Cultures (KCTC; Daejeon, South Korea; https://kctc.kribb.re.kr/) and the Korean Agricultural Culture Collection (KACC; Jeonju, South Korea; http://genebank.rda.go.kr/) (Table 7). All reference strains were cultured in Lactobacilli MRS Broth (Difco, Becton & Dickinson, Sparks, MD, USA) at 30°C for 48 h under anaerobic conditions. The probiotic and dairy products tested in this study were obtained from various markets around the world (South Korea, United States, and Canada). The samples used in this study included 19 probiotic products (10 capsule-form pharmaceuticals and 9 powder-form food supplements) and 12 dairy products manufactured by 19 different companies. All products were labeled with bacterial species or LAB compounds.
All Lactobacillus reference strains were grown in MRS broth at 30°C for 48 h under anaerobic conditions. The cultured cells were harvested by centrifugation at 13,600 × g for 5 min, after which the supernatant was removed. Genomic DNA was extracted using a bacterial genomic DNA extraction kit (Intron Biotechnology, Seongnam, South Korea) according to the manufacturer’s instructions. Total genomic DNA from the probiotic and dairy products was extracted using a DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the method described in a previous study . DNA concentration and purity were determined by absorbance using a MaestroNano® spectrophotometer (Maestrogen, Las Vegas, NV, USA).
Identification of Lactobacillus species-specific regions and primer designs
In total, 180 genome sequences, which contain 37 Lactobacillus species, were obtained from the National Center for Biotechnology Information (NCBI; ftp://ftp.ncbi.nlm.nih.gov/genomes/) database (Additional file 1: Table S1). The 16S–23S rRNA regions, including the intergenic spacer regions, of 180 strains were extracted from the Lactobacillus genomes using a script written in the Python language, and the extracted regions were aligned using the Geneious program ver. 11.1.2 (Biomatters Limited, Auckland, New Zealand). According to the alignment results, primer pairs were designed on the basis of species-specific sequences in the 16S–23S rRNA gene. Some Lactobacillus species are difficult to distinguish at the species level because of the high degree of similarity in their 16S–23S rRNA gene sequences. For these species, we have developed species-specific primer pairs from unique genes that exist only in the target species obtained through comparative genomic analysis.
The genome sequences of target species were blasted against the genome of target species using the UBLAST function of USEARCH program ver. 9.0 , with 80% cutoff identity to obtain genes with high similarity . The genes that showed a significant match with the genomes of all target species were considered as core genes of target species. Those genes were then blasted against all of the Lactobacillus genomes except the target species using the UBLAST function of USEARCH program with default parameter settings of 50% cutoff identity . Genes that found no match to all genomes of the non-target species were identified as potential unique genes. The identified potential unique genes were verified using the Basic Local Alignment Search Tool (BLAST) for 52,478,804 sequences including Lactobacillus genomes. Also, it was confirmed whether the unique genes exist in the genome sequences of reference strains using USEARCH program. The genes were confirmed to be unique genes in the species level and found all in the target species used in this study. The species-specific primer pairs were designed based on these genes. To verify the presence of genomic DNA from Lactobacillus species, primer pairs were designed from the conserved regions of 37 Lactobacillus species in the 16S rRNA gene sequence and used as an internal positive control (IPC). All primer pairs were designed using Primer Designer (Scientific and Educational Software, Durham, NC, USA) and synthesized by Bionics Co. Ltd. (Seoul, South Korea).
Specificity of species-specific primer pairs
PCR assays were performed to confirm the specificity of the designed species-specific primer pairs. The specificity was evaluated using 37 Lactobacillus reference strains. PCR products were amplified using the following conditions in a thermocycler (Astec, Fukuoka, Japan): 94°C for 10 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 72°C for 5 min. The 25 µL reaction mixtures contained 20 ng of template DNA of a Lactobacillus reference strain, 0.5 unit of Taq DNA polymerase (TaKaRa BIO Inc., Tokyo, Japan), and species-specific primer pairs. The optimal concentration of each species-specific primer pair obtained from the experiments is shown in Table 1. The amplification products were confirmed by electrophoresis on a 2% agarose gel, and the product bands were visualized under a UV transilluminator (Vilber Lourmat, Marne La Vallee, France).
Development of real-time PCR assay
In this study, we developed the real-time PCR assay that allows each primer pair to run independently to cover each full assay using one primer pair in each well and 37 wells. The real-time PCR assays were performed on the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following conditions: 95°C for 2 min, followed by 30 cycles of 95°C for 5 s and 60°C for 30 s. The melting curve data were generated using one cycle of 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. The amplification mixture with a final volume of 20 µL for real-time PCR assays included 2X LeGene SB-Green Real-Time PCR Master Mix (LeGene Biosciences, San Diego, CA, USA), template DNA, and species-specific primer pairs at optimal concentrations shown in Table 1. To evaluate the analytical accuracy of the real-time PCR assay, a standard curve was constructed using serial dilutions (50 to 0.005 ng) of genomic DNA from Lactobacillus reference strains in triplicate. The specificities of the species-specific primer pairs were tested using 20 ng of DNA extracted from 37 Lactobacillus reference strains. Real-time PCR amplifications of IPC were also confirmed with 37 Lactobacillus reference strains. The results of the real-time PCR were confirmed using 7500 Software V2.3 (Applied Biosystems).
Application of the developed real-time PCR assay in probiotic and dairy products
We designed a validation test to detect 37 Lactobacillus species with real-time PCR in a single reaction using primer pairs. Each well of a reaction plate contained each primer pair and IPC for the simultaneous detection of 37 Lactobacillus species (Additional file 2: Fig. S1). Briefly, 20 ng of product DNA and 2X Master Mix (LeGene Biosciences) were added to each well of the reaction plate containing species-specific primers. Then, real-time PCR was performed in the 7500 Real-Time PCR system (Applied Biosystems). The real-time PCR conditions were similar to those described in “Development of real-time PCR assay” section. Our method included one primer pair in each well, so 37 wells were used for the full assay of each product sample. Therefore, for all products, including mixed samples, the real-time PCR results determined that the corresponding species was included in the product when amplified in a well containing specific primer pair.