Abstract: To achieve rapid and accurate quantification of viable Streptococcus thermophilus cells, this study developed an innovative detection method based on flow cytometry (FCM) by the combined use of a novel oligonucleotide probe (NOP) and propidium monoazide (PMA). Upon photoactivation, PMA formed irreversible covalent crosslinks with DNA in dead cells, which resulted in red fluorescence in dead cells, while S. thermophilus cells were specifically hybridized with the probe, exhibiting green fluorescence. Viable cells were then quantified by flow cytometry. This method exhibited high specificity and enabled accurate identification and quantification of viable S. thermophilus cells. The limit of quantification was 8.3 × 104 cells/g, with a linear dynamic range of 104–108 cells/g, demonstrating good correlation with the conventional plate counting method. The relative standard deviation (RSD) for repeatability was determined to be 4.68%, reflecting excellent precision. The total detection time was 2 hours, which was significantly shorter compared with the plate counting method. Recoveries from spiked yoghurt samples ranged from 91.61% to 106.17%, and the method was validated for the detection of S. thermophilus in commercially available yoghurts across multiple brands. In conclusion, the PMA-NOP-FCM method offers a highly efficient and precise tool for monitoring microbial viability in industrial yoghurt production, holding great practical significance for yoghurt quality control.

Key words: flow cytometry; Streptococcus thermophilus; oligonucleotide probe; viable bacteria detection; yoghurt