Modified PMA-qPCR Method for Rapid Quantification of Viable Lactobacillus spp. in Fermented Dairy Products

Quantification of viable lactic acid bacteria is an important quality index of fermented dairy products, but the current detection method, which is plate counting, is time-consuming. This study describes a modified PMA-qPCR assay that allows the detection and enumeration of total viable Lactobacillus spp . in commercial fermented milk products. Here, a new pair of genus-specific primers and minor groove binder probes was designed based on the 16S rRNA gene sequences of 24 reference strains, 13 of which were from different Lactobacillus species commonly used in commercial fermented dairy products. Subsequently, compared with the heat killing method, the 20-cycle homogenization method was chosen for initial PMA-qPCR optimization. Standard curves were prepared, and the efficiency obtained was 97% ( R 2 = 0.992), demonstrating that this PMA-qPCR method was feasible and effective. Additionally, the viable Lactobacillus spp. detection range was 10 3 –10 9 CFU/mL, which meets the detection requirements in fermented dairy products. Furthermore, total number of viable Lactobacillus spp. in six different commercial fermented dairy products was estimated by three individual methods (plate count in MRS agar, qPCR, and PMA-qPCR). Comparison of results obtained by PMA-qPCR and plate counts was similar, and both decreased accordingly, showing a good correlation with the decline of strain vitality, 30 days after the expiration date. The PMA-qPCR procedure established in this paper allows the quantification of viable Lactobacillus spp. in approximately 3 h, greatly improving detection efficiency, and has broad application prospects in the testing of fermented dairy products.