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ORIGINAL RESEARCH article

Front. Microbiol.
Sec. Food Microbiology
Volume 15 - 2024 | doi: 10.3389/fmicb.2024.1456274
This article is part of the Research Topic Emerging Technologies for Viability Enumeration of Live Microorganisms View all 11 articles

Development and Validation of a PMA-qPCR Method for Accurate Quantification of Viable Lacticaseibacillus paracasei in Probiotics

Provisionally accepted
Lizheng Guo Lizheng Guo 1Xiaolei ZE Xiaolei ZE 2Yingxin Jiao Yingxin Jiao 1*Chengyu Song Chengyu Song 1*Xi Zhao Xi Zhao 2*Zhiquan Song Zhiquan Song 1*Shuaicheng Mu Shuaicheng Mu 1*Yiru Liu Yiru Liu 1*Yuanyuan Ge Yuanyuan Ge 1*Yu Jing Yu Jing 1*Su Yao Su Yao 1*
  • 1 China National Research Institute of Food and Fermentation, Beijing, China
  • 2 Microbiome Research and Application Center, BYHEALTH Institute of Nutrition & Health, Guangzhou, China

The final, formatted version of the article will be published soon.

    The effectiveness of probiotic products hinges on the viability and precise quantification of probiotic strains. This study addresses this crucial requirement by developing and validating a precise propidium monoazide combination with quantitative polymerase chain reaction (PMA-qPCR) method for quantifying viable Lacticaseibacillus paracasei in probiotic formulations. Initially, species-specific primers were meticulously designed based on core genes from the whole-genome sequence (WGS) of L. paracasei, and they underwent rigorous validation against 462 WGSs, 25 target strains, and 37 non-target strains across various taxonomic levels, ensuring extensive inclusivity and exclusivity. Subsequently, optimal PMA treatment conditions were established using 25 different L. paracasei strains to effectively inhibit dead cell DNA amplification while preserving viable cells. The developed method exhibited a robust linear relationship (R² = 0.994) between cycle threshold (Cq) values and viable cell numbers ranging from 10³ to 10⁸ CFU/mL, with an impressive amplification efficiency of 104.48% and a quantification limit of 7.30 × 10³ CFU/mL. Accuracy assessments revealed biases within ±0.5 Log10 units, while Bland-Altman analysis demonstrated a mean bias of 0.058 Log10, with 95% confidence limits of -0.366 to 0.482 Log10. Furthermore, statistical analysis (P = 0.76) indicated no significant differences between theoretical and measured values. This validated PMA-qPCR method serves as a robust and accurate tool for quantifying viable L. paracasei in various sample matrices, including pure cultures, probiotics as food ingredients, and composite probiotic products, thereby enhancing probiotic product quality assurance and contributing to consumer safety and regulatory compliance.

    Keywords: Probiotics, Lacticaseibacillus paracasei, PMA-qPCR, IDENTIFICATION, Viable cell quantification, Method validation

    Received: 28 Jun 2024; Accepted: 23 Jul 2024.

    Copyright: © 2024 Guo, ZE, Jiao, Song, Zhao, Song, Mu, Liu, Ge, Jing and Yao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Yingxin Jiao, China National Research Institute of Food and Fermentation, Beijing, China
    Chengyu Song, China National Research Institute of Food and Fermentation, Beijing, China
    Xi Zhao, Microbiome Research and Application Center, BYHEALTH Institute of Nutrition & Health, Guangzhou, China
    Zhiquan Song, China National Research Institute of Food and Fermentation, Beijing, China
    Shuaicheng Mu, China National Research Institute of Food and Fermentation, Beijing, China
    Yiru Liu, China National Research Institute of Food and Fermentation, Beijing, China
    Yuanyuan Ge, China National Research Institute of Food and Fermentation, Beijing, China
    Yu Jing, China National Research Institute of Food and Fermentation, Beijing, China
    Su Yao, China National Research Institute of Food and Fermentation, Beijing, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.