Abstract
The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.
Highlights
Total enumeration of viable bacteria is a key metric in probiotic industrial science used to ensure strain-specific health benefits, per the definition of probiotics (Hill et al, 2014)
The practice of obtaining colony forming units (CFU) by plating has been used since the late 19th century and is the current standard for enumeration in the probiotic industry (Davis, 2014) with publications from the International Organization of Standardization (ISO) and United States Pharmacopeia (USP) (United States Pharmacopeial Convention, 2015)
Various primer concentrations were tested for optimal amplification, with 0.6 mM forward and 0.9 nmM reverse primers preforming the best. quantitative PCR (qPCR) was used to assess the efficiency of the primer set on its ability to detect DNA extracted from pure culture
Summary
Total enumeration of viable bacteria is a key metric in probiotic industrial science used to ensure strain-specific health benefits, per the definition of probiotics (Hill et al, 2014) This has typically been assessed by the microbial growth on media that selects for target phenotypic and metabolic characteristics. Generation sequencing (NGS) efforts have estimated relative abundances of probiotics based on 16S rRNA gene profiling (Morovic et al, 2016) and metagenomics analysis (Patro et al, 2016). These methods all quantify microbes using technologies that have limitations in crucial criteria
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