Droplet digital PCR method for the absolute quantitative detection and monitoring of Lacticaseibacillus casei

Droplet digital polymerase chain reaction (ddPCR) is an emerging molecular detection assay that provides an absolute quantification of targets. Despite its emerging applications in the detection of food microorganisms, there are limited reports of its use for the monitoring of microorganisms utilized as starters in the dairy industry. This study investigated the applicability of ddPCR as a detection platform for Lacticaseibacillus casei, a probiotic found in fermented foods and exerts beneficial effects on human health. In addition, this study compared the performance of ddPCR with that of real-time PCR. The ddPCR targeting the haloacid dehalogenase-like hydrolase (LBCZ_1793) exhibited high specificity against 102 nontarget bacteria, including Lacticaseibacillus species that is very closely related to L. casei. The ddPCR exhibited high linearity and efficiency within the quantitation range (10⁵-10⁰ CFU/ml), with the limit of detection being 10⁰ CFU/ml. The ddPCR also demonstrated a higher sensitivity than real-time PCR in detecting low bacterial concentration in spiked milk samples. Furthermore, it provided an accurate absolute quantification of the concentration of L. casei, without the need for standard calibration curves. This study demonstrated that ddPCR is a useful method for monitoring starter cultures in dairy fermentations and detecting L. casei in foods.