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Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains
Sarah J Z Hansen1, Peipei Tang1, Anthony Kiefer1
1DuPont Nutrition & Biosciences, Madison, WI, United States.
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.
Insights
Droplet digital PCR (ddPCR) offers a faster, more precise method for enumerating probiotic microorganisms compared to traditional plate counts and flow cytometry. This advanced technique provides strain-specific counts, crucial for accurate probiotic dosing and quality control.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Traditional microbiological enumeration methods for probiotics are time-consuming, variable (up to 35%), and hinder precise dose determination.
- Accurate probiotic enumeration is critical for clinical research, microbiome science, and ensuring consistent consumer product quality.
- Existing methods like flow cytometry offer improvements but face adoption challenges due to implementation complexities.
Purpose of the Study:
- To compare the accuracy and efficiency of droplet digital PCR (ddPCR) against traditional plate counts and flow cytometry for probiotic enumeration.
- To evaluate ddPCR's potential as a next-generation method for precise probiotic quantification and strain profiling.
- To assess the correlation of ddPCR results with established enumeration techniques.
Main Methods:
- Enumeration of probiotic microorganisms using standard plate counts.
- Flow cytometry analysis for microbial cell quantification.
- Droplet digital PCR (ddPCR) with strain-specific primer design for targeted enumeration.
- Comparative analysis of results from all three methods.
Main Results:
- ddPCR demonstrated faster time-to-results compared to plate counts.
- ddPCR exhibited significantly lower variability than both plate counts and flow cytometry.
- ddPCR results showed a positive correlation with both plate count and flow cytometry data.
- ddPCR enabled strain-specific quantification, allowing for detailed profiling.
Conclusions:
- ddPCR is a viable and superior next-generation method for probiotic enumeration.
- The precision and speed of ddPCR support accurate probiotic dosing and quality assurance.
- ddPCR's strain-specific capabilities are valuable for microbiome research and product characterization.
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