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Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR
Lizheng Guo1, Xiaolei Ze2, Huifen Feng1
1China National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, China.
Frontiers in Microbiology
|February 1, 2024
Summary
Quantifying viable Lacticaseibacillus rhamnosus in probiotics is difficult. This study developed a reliable propidium monoazide quantitative PCR (PMA-qPCR) method for accurate detection and enumeration of live L. rhamnosus in probiotic products.
Area of Science:
- Microbiology
- Molecular Biology
- Food Science
Background:
- Accurate quantification of viable bacteria in complex probiotic formulations presents significant challenges.
- Molecular methods like propidium monoazide (PMA) combined with quantitative PCR (qPCR) are emerging for precise viable cell enumeration.
- Species- and strain-level identification of probiotics in commercial products requires robust and validated methodologies.
Purpose of the Study:
- To establish and validate a robust PMA-qPCR method for the accurate detection and quantification of viable Lacticaseibacillus rhamnosus.
- To systematically assess key performance metrics including relative trueness, accuracy, limit of quantification, linearity, and range.
- To confirm the specificity and inclusivity of the developed method for L. rhamnosus.
Main Methods:
- Development of a propidium monoazide (PMA) treatment protocol optimized for discriminating between live and dead Lacticaseibacillus rhamnosus.
- Quantitative PCR (qPCR) assay design and validation using specific primers for L. rhamnosus.
- Systematic validation of the PMA-qPCR method using inclusivity/exclusivity panels, standard curves, and application to diverse probiotic samples.
Main Results:
- The developed PMA-qPCR method demonstrated high specificity for L. rhamnosus, confirmed by inclusivity and exclusivity testing.
- PMA treatment effectively differentiated between live and dead bacterial cells across various L. rhamnosus strains.
- The method accurately quantified viable L. rhamnosus from 10^3 to 10^8 CFU/mL in pure cultures and complex probiotic products, showing good agreement with theoretical values.
Conclusions:
- A validated PMA-qPCR method provides a reliable tool for the accurate quantification of viable Lacticaseibacillus rhamnosus.
- This method offers potential for industrial applications in the quality control and regulatory supervision of probiotic products.
- The established protocol effectively addresses the challenge of viable bacterial enumeration in complex probiotic matrices.
