短双歧杆菌靶向适配体荧光微球的制备及其在流式检测益生菌制品中的应用

doi:10.7506/spkx1002-6630-20251223-192

食品科学 ›› 2026, Vol. 47 ›› Issue (12): 356-364.doi: 10.7506/spkx1002-6630-20251223-192

• 安全检测 • 上一篇

短双歧杆菌靶向适配体荧光微球的制备及其在流式检测益生菌制品中的应用

郭宝姣，曲天铭，赵连慧，李丁，刘赛钦，陈颖，王娉，武运

（1.新疆农业大学食品科学与药学学院，新疆乌鲁木齐 830000；2.中国质量检验检测科学研究院，北京 100076）

发布日期:2026-07-08
基金资助:
中国质量检验检测科学研究院基本科研业务费项目（2025JK062）；国家市场监督管理总局科技创新人才计划项目（QNBJ202327）

Preparation and Application of Aptamer-Based Fluorescent Microspheres in Flow Cytometric Detection of Bifidobacterium breve in Probiotic Products

GUO Baojiao, QU Tianming, ZHAO Lianhui, LI Ding, LIU Saiqin, CHEN Ying, WANG Ping, WU Yun

(1. College of Food Science and Pharmacy, Xinjiang Agricultural University, ürümqi 830000, China; 2. Chinese Academy of Quality and Inspection & Testing, Beijing 100076, China)

Published:2026-07-08

摘要/Abstract

摘要： 目的：以核酸适配体作为捕获探针、聚苯乙烯荧光微球作为信号探针，构建一种基于流式细胞术的益生菌制品中短双歧杆菌活/死菌的快速计数方法。方法：通过生物素-链霉亲和素介导的非共价结合，将短双歧杆菌核酸适配体偶联于聚苯乙烯荧光微球，制备靶向短双歧杆菌的适配体荧光微球。使用碘化丙啶对样本进行荧光染色，通过优化反应条件、染色剂浓度、荧光通道阈值等，建立流式细胞仪检测方法，并与国标方法进行比较。结果：当适配体质量浓度为1 mg/mL、荧光微球浓度为1 000 nmol/L、孵育温度为37 ℃、孵育时间为45 min时，适配体-荧光微球结合效率达到峰值。该方法在102～108 CFU/mL浓度范围内对短双歧杆菌的荧光信号强度与菌浓度对数值呈显著线性相关（R2＝0.991 9），检测限（limit of detection，LOD）为102 CFU/mL，优于常规的流式计数法（LOD＝1×103 CFU/mL）。该方法检测加标样品结果与平板计数法结果无显著差异（P＞0.05），方法回收率为98.00%～101.33%，实际样品检测相对标准偏差≤7.53%，且检测全程用时小于2 h，效率较平板计数方法效率提升超20 倍。结论：本研究建立的基于流式细胞术的适配体-微球方法具有快速、灵敏、准确的特点，能够有效应用于益生菌产品中短双歧杆菌的定量检测，为短双歧杆菌在食品加工、肠道微生态研究等领域的精准定量提供可靠技术。

关键词: 短双歧杆菌；靶向检测；核酸适配体；荧光定量微球；流式细胞术

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

中图分类号:

TS207.4

郭宝姣，曲天铭，赵连慧，李丁，刘赛钦，陈颖，王娉，武运. 短双歧杆菌靶向适配体荧光微球的制备及其在流式检测益生菌制品中的应用[J]. 食品科学, 2026, 47(12): 356-364 https://doi.org/10.7506/spkx1002-6630-20251223-192

GUO Baojiao, QU Tianming, ZHAO Lianhui, LI Ding, LIU Saiqin, CHEN Ying, WANG Ping, WU Yun. Preparation and Application of Aptamer-Based Fluorescent Microspheres in Flow Cytometric Detection of Bifidobacterium breve in Probiotic Products[J]. FOOD SCIENCE, 2026, 47(12): 356-364 https://doi.org/10.7506/spkx1002-6630-20251223-192

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短双歧杆菌靶向适配体荧光微球的制备及其在流式检测益生菌制品中的应用

Preparation and Application of Aptamer-Based Fluorescent Microspheres in Flow Cytometric Detection of Bifidobacterium breve in Probiotic Products

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