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

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.