Abstract: A droplet digital polymerase chain reaction (ddPCR) method for the quantitative detection Acetobacter aceti in fermented milk was established. Specific primers and probes were designed according to the internally transcribed spacer (ITS) gene sequence of Acetobacter aceti, and annealing temperature was optimized. The specificity of the method was verified by applying it on various strains, the limit of detection (LOD) was determined for artificially inoculated Acetobacter aceti, and the absolute quantification was systematically investigated by comparing the results of ddPCR and the counting results. The experimental results showed that the optimal annealing temperature was 54.6 ℃, the method had strong specificity and high sensitivity, and the LOD was 7.2 × 101 CFU/mL. The quantitative deviation rate was 23.73%. This method can meet the demand for quantitative detection of Acetobacter aceti in fermented milk.

Key words: droplet digital polymerase chain reaction; fermented milk; Acetobacter aceti; quantitative detection

摘要： 建立一种微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）定量检测发酵乳中醋化醋杆菌的技术。根据醋化醋杆菌ITS基因序列设计特异性引物和探针，通过反应温度筛选优化ddPCR扩增条件，通过多种菌株扩增验证方法特异性，通过人工添加醋化醋杆菌验证方法检出限，通过ddPCR法实测结果和计数法测定结果之间的比较对其绝对定量进行系统性研究。结果表明：建立的发酵乳中醋化醋杆菌ddPCR定量检测方法，反应条件中最适退火温度为54.6?℃，方法特异性强，检出限为7.2×101?CFU/mL，灵敏度高，定量偏差率为23.73%。该方法可以满足发酵乳中醋化醋杆菌定量检测需求。

关键词: 微滴式数字聚合酶链式反应技术；发酵乳；醋化醋杆菌；定量检测