双重实时荧光聚合酶链式反应技术检测非活菌型发酵乳中乳酸菌

Detection of lactic acid bacteria in non-viable fermented milk by duplex real-time fluorescent polymerase chain reaction technology

中文关键词:  非活菌型  发酵乳  双重实时荧光PCR  乳酸菌

英文关键词:non-viable fermented milk  duplex real-time fluorescent polymerase chain reaction  lactic acid bacteria

中文摘要:

      目的 建立双重实时荧光聚合酶链式反应(real-time fluorescent polymerase chain reaction, RT-PCR)方法检测非活菌型发酵乳中唾液链球菌亚种和德氏乳杆菌保加利亚亚种的分析方法。方法 优化双重RT-PCR法反应体系中两个目标引物探针的比例、引物探针的浓度、Mg2+的浓度以及反应程序中退火温度, 验证该方法的特异性和重复性, 最后对市场上购买的实际样本进行目标菌的检测。结果 20 μL反应体系中两个目标引物探针的比例为1:1、上下游引物(10 μmol/L)各0.30 μL、探针(10 μmol/L)各0.150 μL、Mg2+的浓度为3.0 mmol/L以及退火温度为60 ℃扩增效果较好, 24批次市场上购买的实际样本均检出唾液链球菌亚种和德氏乳杆菌保加利亚亚种。结论 本研究建立的方法可快速、准确检测出非活菌型发酵乳中是否含有唾液链球菌亚种和德氏乳杆菌保加利亚亚种, 为食品真实性监管提供有效的技术支撑。

英文摘要:

      Objective To establish a duplex real-time fluorescent polymerase chain reaction (RT-PCR) method for detecting Streptococcus salivarius subspecies and Lactobacillus delbrueckii subspecies bulgaricus in non-viable fermented milk. Methods The optimal reaction system of duplex RT-PCR was optimized by adjusting the ratio of 2 target primers/probes, primer/probe concentrations, Mg2+ concentration and annealing temperature. The specificity and reproducibility of the method were validated. Finally, the target bacteria were detected in the actual samples purchased from the market. Results Optimal amplification was achieved in a 20 μL reaction system containing primer-probes for both targets at a 1:1 ratio, 0.3 μL each of forward and reverse primers (10 μmol/L), 0.15 μL each of probes (10 μmol/L), 3 mmol/L Mg2+ and an annealing temperature of 60 ℃. Both Streptococcus salivarius subspecies and Lactobacillus delbrueckii subspecies bulgaricus were detected in all 24 batches of commercial samples. Conclusion The established method enables rapid and accurate detection of Streptococcus salivarius subspecies and Lactobacillus delbrueckii subspecies bulgaricus in non-viable fermented milk, providing effective technical support for food authenticity supervision.