AUTHOR=Guo Lizheng , Ze Xiaolei , Jiao Yingxin , Song Chengyu , Zhao Xi , Song Zhiquan , Mu Shuaicheng , Liu Yiru , Ge Yuanyuan , Jing Yu , Yao Su 

TITLE=Development and validation of a PMA-qPCR method for accurate quantification of viable Lacticaseibacillus paracasei in probiotics

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1456274

DOI=10.3389/fmicb.2024.1456274

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Lacticaseibacillus paracasei</italic> in probiotic formulations. Initially, species-specific primers were meticulously designed based on core genes from the whole-genome sequence (WGS) of <italic>L. paracasei</italic>, 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 <italic>L. paracasei</italic> strains to effectively inhibit dead cell DNA amplification while preserving viable cells. The developed method exhibited a robust linear relationship (<italic>R</italic><sup>2</sup> = 0.994) between cycle threshold (C<sub>q</sub>) values and viable cell numbers ranging from 10<sup>3</sup> to 10<sup>8</sup> CFU/mL, with an impressive amplification efficiency of 104.48% and a quantification limit of 7.30 × 10<sup>3</sup> CFU/mL. Accuracy assessments revealed biases within ±0.5 Log<sub>10</sub> units, while Bland–Altman analysis demonstrated a mean bias of 0.058 Log<sub>10</sub>, with 95% confidence limits of −0.366 to 0.482 Log<sub>10</sub>. Furthermore, statistical analysis (<italic>p</italic> = 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 <italic>L. paracasei</italic> 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.</p>