Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains

Abstract

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability-up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

Optimization of Viability Treatment Essential for Accurate Droplet Digital PCR Enumeration of Probiotics

“…Additionally, these assays were assessed for linearity using three commercial lots diluted over a 2-log concentration gradient and were found to have excellent linear relationship. In agreement with previous literature, we have demonstrated the ability of viability dyes (PMA, EMA, PEMAX, and PE51), paired with PCR based methods (qPCR, cdPCR, and ddPCR) to meet this criterion (Hansen et al, 2018;Hansen et al, 2020;Meng et al, 2010;Morovic et al, 2018).…”

“…Strain specific primer and probe assays were first reported by Hansen et al (2020). GroEL assays designed for this study were in the same location on the GroEL gene for Bi-07 and La-14 and to have similar amplicon length, GC content, and Tm to strain specific counterparts (Figure 1 and Table 1).…”

“…lactis Bl-04 and Lactobacillus acidophilus NCFM were successfully differentiated against other strains of the same species and selectively enumerated when multiplexed (Hansen et al, 2018). Additionally, ddPCR was shown to enumerate several commercial probiotic strains with a high correlation to the gold standard of plate counts, and with the additional advantage of generating faster (4-8 vs. 24-72 h) and more accurate (1-3% vs. 12-20% CV) results (Hansen et al, 2020).…”

“…We have previously shown that dPCR is a viable alternative to plate counts for the rapid, accurate and strain-specific enumeration of commercial probiotics ( Hansen et al, 2018 , 2020 ). In this study, we evaluate and demonstrate the need for optimizing the concentration of the dual stain PMA-EMA, here after referred to as PE51, based on the bacterial genus to be enumerated and on the gene to be targeted by viability droplet digital PCR (v-ddPCR).…”

The Development of High-Quality Multispecies Probiotic Formulations: From Bench to Market

“…Strain-specific polymerase chain reaction (PCR) assays can be developed with the use of strain-specific PCR primer sets and probes that target unique insertions/deletions or single-nucleotide polymorphisms in DNA sequences based on which they are able to even distinguish phylogenetically similar strains. In addition, the field is currently developing methods to quantify viable cells using a PCR-based method in combination with live-dead staining methods [ 55 , 56 ]. These methods are based on the approach that prior to DNA extraction and amplification, samples are treated with a viability dye, a molecule that selectively enters cells with damaged membranes and intercalates into their DNA.…”