Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method

Abstract

The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR C_q values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 10³ to 10⁸ CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.

Keywords: Lacticaseibacillus rhamnosus; PMA-qPCR method; identification; probiotics; validation; viable cell quantification.