Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

Abstract

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

Keywords: Bifidobacterium animalis subsp. lactis; authentication; locked nucleic acid probe; probe-based; probiotics; real-time PCR; strain-specific.

FIGURE 1

Multiple sequence alignment from NCBI multiple sequence alignment viewer 1.20.1. The amplicon sequence of (A) Bifidobacterium animalis subsp. lactis Bi-07 and (B) B. animalis subsp. lactis DSM 15954 assays were searched on GenBank using the blastn function against the Nucleotide collection database to find matches in all publicly available genome sequences. The probe sequence is in a blue box and the locked nucleic acid bases are in an orange box. The single nucleotide polymorphism (SNP) identified in Bi-07 was unique to strain Bi-07 compared to all other Bifidobacterium animalis subsp. lactis strains deposited in GenBank, while the SNP identified in DSM 15954 was unique to all strains except IDCC4301, BF052, RH, and i797, as of August 2021.

FIGURE 2

Evaluating the specificity of the developed strain-specific assays. (A) Bifidobacterium animalis subsp. lactis DSM 15954 assay and (B) Bifidobacterium animalis subsp. lactis Bi-07 assay. Numbers of target samples tested were 13 and 25 for DSM 15954 and Bi-07 assays, respectively. Numbers of non-target samples tested was 22 for each assay. Each sample was tested in triplicate.

FIGURE 3

Evaluating the analytical sensitivity and efficiency of the developed strain-specific assays. (A) Bifidobacterium animalis subsp. lactis DSM 15954 assay and (B) Bifidobacterium animalis subsp. lactis Bi-07 assay. Three 10-fold dilution series were prepared from three starting DNA concentrations (10 ng/μl, 5 ng/μl, and 2 ng/μl). Each dilution series was prepared at five dilution points and each dilution was tested in triplicate. Limits of detection were 5 pg, corresponding to 2388 target copies, and 0.5 pg, corresponding to 239 target copies, for DSM 15954 and Bi-07 assays, respectively.

FIGURE 4

Evaluating repeatability and reproducibility of the developed strain-specific assays. (A) Bifidobacterium animalis subsp. lactis DSM 15954 assay and (B) Bifidobacterium animalis subsp. lactis Bi-07 assay. Five samples at three different DNA concentrations (1 ng/μl, 0.1 ng/μl and 0.01 ng/μl) were used. Each assay was repeated on a different day to evaluate repeatability and was repeated on a different real-time PCR platform (bCUBE and QuantStudio 5) to evaluate reproducibility.

FIGURE 5

Application of the developed strain-specific assays in various product matrices. Bifidobacterium animalis subsp. lactis DSM 15954 (A) and Bi-07 (B) were added to a variety of products and food matrices before DNA extraction. The Cq values from samples with matrices were compared to the Cq value of the culture only control to assess possible PCR inhibition. Shown are bars representing the mean with standard error of the mean (SEM). No significant difference in Cq values was observed between the control sample and any of the sample matrices in both assays (Dunn’s multiple comparisons test), indicating no inhibitory effect.