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.
Highlights
Recent years have witnessed a significant increase in scientific investigations of probiotics with over 20,000 publications as of February 2019 (Reid et al, 2019)
A recent review by a probiotic expert panel suggested that strains are defined by a single genetic sequence, and that they can be distinguished by even single nucleotide polymorphisms (SNP) (Jackson et al, 2019)
locked nucleic acids (LNA) oligos were implemented in hydrolysis probe-based qPCR methods for simple, fast, and sensitive identification of the two B. animalis subsp. lactis strains
Summary
Recent years have witnessed a significant increase in scientific investigations of probiotics with over 20,000 publications as of February 2019 (Reid et al, 2019). The rapid growth in scientific research and in the market size of probiotics was accompanied by reports on non-compliance and fraud in probiotic products (Morovic et al, 2016; Patro et al, 2016; Kolacek et al, 2017; Shehata and Newmaster, 2020b,c). Relevant to correct probiotic characterization is proper strain identification and naming (Binda et al, 2020). The importance of identification to strain level is becoming increasingly recognized since probiotic health benefits are strain specific, unless otherwise proven (Klein et al, 2010; McFarland et al, 2018). Reliable and highly specific strain identification methodologies are an important component in probiotic authentication and quality assessment
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