叠氮溴化丙锭-qPCR法定量检测植物乳杆菌

Quantitative detection of Lactobacillus plantarum by propidium monoazide-qPCR

目的将叠氮溴化丙锭(propidium monoazide,PMA)与实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qPCR)相结合,建立一种活性植物乳杆菌定量检测的PMA-qPCR方法。方法首先进行引物探针特异性验证,优化PMA反应条件,建立标准曲线,验证灵敏度,然后将该方法用于检测人工添加奶粉、米粉、酸奶样品以及冻干样品中的植物乳杆菌。结果最佳PMA浓度为2μg/mL,最佳曝光时间为10 min,建立的PMA-qPCR方法可以快速、高效、特异地检测植物乳杆菌,利用细菌纯培养物与对应Ct值建立标准曲线,可以看出纯培养物浓度与Ct值之间存在对应线性关系,相关系数(r^(2))为0.9992,最低检测限为15拷贝/反应体系,人工添加样品以及冻干样品检测中PMA-qPCR和平皿计数法结果的对数值进行配对t检验,显示在不同的人工添加样本以及冻干样品中均无显著性差异(P>0.05)。结论本研究建立的PMA-qPCR检测方法能快速、准确、特异、灵敏地定量检测活性植物乳杆菌。

Objective To establish a quantitative assay method to detect Lactobacillus plantarum based on using propidium monoazide(PMA) combined with real-time fluorescence quantitative polymerase chain reaction(qPCR). Methods The specificity of the primer probe was verified, the PMA reaction condition was optimized, and the standard curve was established to verify the sensitivity. Secondly, the method was applied to the detection of Lactobacillus plantarum in artificially added milk powder, rice flour, yogurt samples and lyophilized samples. Results The optimal PMA concentration was 2 μg/mL, and the optimal exposure time was 10 min. The established PMA-qPCR method could detect Lactobacillus plantarum rapidly, efficiently and specifically. The standard curve was established based on the pure bacterial culture and the corresponding Ct value. It could be seen that there was a corresponding linear relationship between the pure culture concentration and Ct value, with the correlation coefficient(r^(2)) of 0.9992 and the minimum detection limit of 15 copies/reaction system. The paired t-test of the paired values of PMA-qPCR and plate counting in the detection of manually added samples and lyophilized samples showed no significant difference among the different manually added samples and lyophilized samples(P>0.05). Conclusion This PMA-qPCR detection method can quickly, accurately, specifically and sensitively quantitatively detect active Lactobacillus plantarum.