Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China; College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China; Hebei Key Laboratory of Analysis and Control for Zoonotic Pathogenic Microorganism, Hebei Agricultural University, Baoding 071001, China.
Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China.
J Dairy Sci. 2024 Oct;107(10):7678-7690. doi: 10.3168/jds.2024-24876. Epub 2024 Jun 20.
Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA)-antibiotic fluorescence in situ hybridization (AFISH)-flow cytometry (FC) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and nonviable cells. The PMA-AFISH-FC method, including sample pretreatment, CM treatment, dual staining, and FC analysis, required approximately 2 h and was found to be better than the current methods. This is the first study to implement FC combined with PMA and an oligonucleotide probe for detecting Bifidobacterium.
由于双歧杆菌对人体健康有益，因此常被添加到奶粉中。准确量化活菌双歧杆菌对于评估奶粉的治疗效果至关重要。在这项研究中，我们引入了一种新的吖啶橙单脒（PMA）-抗生素荧光原位杂交（AFISH）-流式细胞术（FC）方法，可快速准确地量化奶粉中的活菌双歧杆菌细胞。简要地说，用氯霉素（CM）处理双歧杆菌细胞以增加其 rRNA 含量，然后基于 AFISH 技术用 RNA 结合寡核苷酸探针进行染色。然后，用 DNA 结合染料 PMA 将活细胞和死细胞区分开来。PMA-AFISH-FC 方法，包括样品预处理、CM 处理、双重染色和 FC 分析，大约需要 2 小时，并且比目前的方法更好。这是首次实施 FC 与 PMA 和寡核苷酸探针结合检测双歧杆菌的研究。
