Work

Development of a new PMA-qPCR method for quantification of four viable lactic acid bacteria species based on novel molecular markers

Year: 2026

Type: article

Abstract: The recovery and quantification of viable probiotic cells from food samples are essential for verifying product efficacy and quality. The traditional culture methods are time-consuming and laborious. In this study, 998 genomic sequences of LAB were analyzed and four novel species-specific molecular markers were mined for Limosilactobacillus reuteri , Lacticaseibacillus rhamnosus , Lacticaseibacillus paracasei , and Lactobacillus acidophilus . The specificity of the genetic markers was evaluated using 15 LAB species. A new Propidium Monoazide-qPCR (PMA-qPCR) assay was developed to quantify four viable LAB species. The optimal PMA conditions were 40 μmol/L with 30 min of light exposure, 20 μmol/L with 20 min, 30 μmol/L with 20 min, and 30 μmol/L with 25 min for L. reuteri , L. rhamnosus , L. paracasei , and L. acidophilus , respectively. Four quantitative standard curves were established, demonstrating a robust correlation ( R 2 > 0.99) with viable cell counts over an extensive concentration range (10 3 –10 8 CFU/mL). The detection limits were 8.45 × 10 3 CFU/mL, 4.30 × 10 2 CFU/mL, 7.40 × 10 3 CFU/mL, and 1.32 × 10 2 CFU/mL of pure culture for L. reuteri , L. rhamnosus , L. paracasei , and L. acidophilus , respectively. In 59 actual probiotic products, there was no significant difference between the results of PMA-qPCR and the traditional culture method ( P >0.05). The assay can accurately quantify viable bacteria in probiotic products within approximately 3 hours and holds great promise for quality-control applications. • Novel genetic markers were mined for four probiotic species by analyzing 998 LAB genomes. • The primers demonstrated high specificity across 15 different LAB species. • A novel PMA-qPCR assay was developed to quantify viable cells of four LAB species. • The PMA-qPCR assay exhibited good reliability in detecting 59 probiotic products.

Source: LWT

DOI: 10.1016/j.lwt.2026.119064