叠氮溴化丙锭-qPCR定量检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III

Quantitative determination of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus plantarum ST-III in fermented milk by propidium monoazide-qPCR

中文关键词:  叠氮溴化丙锭  实时荧光定量PCR  动物双歧杆菌乳亚种BB-12  植物乳植杆菌ST-Ⅲ

英文关键词:propidium monoazide  real-time fluorescence quantitative polymerase chain reaction  Bifidobacterium animalis subsp. lactis BB-12  Lactiplantibacillus plantarum ST-III

中文摘要:

      目的 将叠氮溴化丙锭(propidium monoazide, PMA)与实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)联合, 建立一种发酵乳体系中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的活菌定量检测方法。方法 首先设计动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的特异性基因序列的引物探针, 验证引物探针特异性, 优化PMA反应条件, 建立标准曲线, 验证灵敏度, 最后定量检测发酵乳样品中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ菌株的数量。结果 动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ经PMA处理受损菌的最佳质量浓度为10 μg/mL, 处理菌液样品的最佳曝光时间为10 min, PMA-qPCR方法能够准确检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ。结论 本研究建立了一种快速、准确PMA-qPCR检测方法, 用于检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ, 为发酵乳产品中益生菌的定量检测提供一定的借鉴意义。

英文摘要:

      Objective To establish a quantitative detection method for the quantitative determination of the viable bacteria number of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk, by combining propidium monoazide (PMA) with real-time fluorescence quantitative polymerase chain reaction (qPCR). Methods Firstly, the specific primer probe of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was designed and verified, the standard curve was established using standard strains, and the reaction conditions of the PMA were optimized. Secondly, the quantities of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in the fermented milk samples were quantitatively detected. Results The optimal mass concentration of damaged bacteria of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was 10 μg/mL, and the optimal exposure time was 10 min. The PMA-qPCR detection method could accurately detect Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk. Conclusion In this study, a rapid and accurate PMA-qPCR detection method is established for the detection of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk system, which provides certain reference significance for the quantitative detection of probiotics in fermented milk products.