Abstract: Objective: Using nucleic acid aptamers as the capture probe and polystyrene fluorescent microspheres as the signal probe, we established a rapid method for counting viable/dead Bifidobacterium breve in probiotic products based on flow cytometry (FCM). Methods: B. breve nucleic acid aptamers were conjugated to fluorescent polystyrene microspheres via biotin-streptavidin mediated non-covalent binding, yielding aptamer-functionalized fluorescent microspheres for B. breve detection. Propidium iodide (PI) was used for fluorescent staining of samples. An FCM method was established by optimizing reaction conditions, staining agent concentration and fluorescence channel thresholds, and it was evaluated against the national standard method. Results: The binding efficiency of aptamer to fluorescent microspheres peaked under the conditions of 1 mg/mL aptamer concentration, 1 000 nmol/L fluorescent microsphere concentration, and incubation at 37 ℃ for 45 min. The fluorescence signal intensity exhibited good linearity with the logarithm of bacterial concentration in the range of 102–108 CFU/mL (R2 = 0.991 9). The limit of detection (LOD) of this method was superior to that of conventional flow cytometry (102 CFU/mL versus 1 × 103 CFU/mL). The results of this method for spiked samples showed no significant difference from those of the plate counting method (P > 0.05). Its recoveries ranged from 98.00% to 101.33%, with relative standard deviation (RSD) ≤ 7.53% for actual samples. Moreover, the total detection time was less than 2 hours, which was more than 20-fold shorter than that of the plate counting method. Conclusion: The FCM method with aptamer-modified fluorescent microspheres is characterized by rapidity, high sensitivity and accuracy. It can be effectively applied to the quantitative detection of B. breve in probiotic products, providing a reliable technical tool for the precise quantification of B. breve in fields such as food processing and intestinal microecology research.

Key words: Bifidobacterium breve; targeted detection; nucleic acid aptamer; fluorescent microspheres; flow cytometry