SYBR Green I染料法实时荧光定量PCR检测发酵乳中的霉菌和酵母

Detection of molds and yeasts in fermented milk by real-time fluorescent quantitative PCR with SYBR Green I dye

中文关键词:  霉菌  酵母  SYBR Green I染料  检测  实时荧光定量PCR  发酵乳

英文关键词:mold  yeast  SYBR Green I dye  detection  real-time fluorescence quantitative PCR  Fermented milk

中文摘要:

      目的 建立一种应用SYBR Green I染料的实时荧光定量PCR方法，从而实现对发酵乳中霉菌和酵母菌的快速检测。 方法 本研究针对霉菌与酵母菌的保守序列设计引物，确定最优反应体系与反应条件。通过熔解曲线与以及其他真菌作为非目标菌株的检测验证该方法的特异性；通过菌悬液的梯度稀释检测确定该方法的灵敏度；通过菌悬液与发酵乳样品混合后进行检测，确定该方法的检出限，并得到标准曲线。 结果 该方法能够特异性的检测出发酵乳中的目标菌株，无交叉反应。该方法检测霉菌、酵母菌的灵敏度均为102 CFU/mL，并且当菌悬液与发酵乳样品混合后，并没有降低该方法的检出限，仍为102 CFU/mL。在106至102 CFU/mL浓度范围内，菌液浓度的对数值与Ct值具有良好的线性关系，可以在市售样品的检测中通过Ct值计算样品中目标菌的含量，更快的判断发酵乳是否被真菌污染。 结论 该方法可用于发酵乳中霉菌和酵母菌的快速检测。为发酵乳的食品安全提供了技术保障。

英文摘要:

      Objective Development of a real-time fluorescence quantitative PCR method applying SYBR Green I dye for rapid detection of molds and yeasts in fermented milk.Methods In this study, primers were respectively designed for the conserved sequences of molds and yeasts, and the optimal reaction system and conditions were determined. The specificity of the method was verified by melting curves and the detection of other fungi as non-target strains; the sensitivity of the method was determined by a gradient dilution assay of the bacterial suspension; determination of the detection limit of the method by mixing the bacterial suspension with fermented milk samples and obtaining the standard curve. Results The method was able to specifically detect the target strains in fermented milk without cross-reactivity. The sensitivity of the method for detecting either molds or yeasts was 102 CFU/mL. When the fungal suspension was mixed with the fermented milk sample, it did not lower the detection limit of the method, which was still 102 CFU/mL. Meanwhile, the logarithmic values of fungal suspension concentrations showed good linear relationship with Ct values in the concentration range of 106 to 102 CFU/mL. It is possible to calculate the amount of target bacteria in the sample by Ct value in the testing of commercially available samples, and to determine more quickly whether fermented milk is contaminated with fungi. Conclusion The method can be used for the rapid detection of molds and yeasts in fermented milk. It provides a technical guarantee for the food safety of fermented milk.