TaqMan-MGB探针荧光定量聚合酶链式反应法检测酸奶制品中动物双歧杆菌BB-12

Determination of animal Bifidobacterium BB-12 in yogurt products by TaqMan-MGB fluorescent quantitative polymerase chain reaction

目的建立一种TaqMan-小沟结合物(minor groove binder,MGB)探针荧光定量聚合酶链式反应法检测酸奶制品中动物双歧杆菌BB-12的分析方法。方法设计动物双歧杆菌BB-12的16SrRNA序列的MGB探针,利用荧光定量聚合酶链式反应(polymerase chain reaction, PCR)技术,构建目标序列的过表达载体,提取质粒进行标准曲线的绘制,最后进行市售酸奶样品检测,定量酸奶中BB-12菌株的数量。结果所有载体标准品均产生显著的荧光增幅,循环阈值(cycle threshold, Ct)介于16~20之间;而以其他酸奶制品添加菌及大肠杆菌样品DNA为模板则无荧光扩增信号。在检测灵敏度方面,BB-12载体可被稳定检出的质量浓度低至0.0000584 ng/μL, Ct值平均数为28.73,即检测灵敏度低至为0.05 pg。绘制的标准曲线线性良好,扩增效率E为0.39,判定系数R^(2)=0.9999。市售酸奶样品检测特异性好、灵敏度高、定量结果准确。结论本研究建立的TaqMan-MGB探针荧光定量PCR检测法可实现在酸奶中对BB-12菌种的检测及定量。

Objective To establish an analytical method for the detection of animal Bifidobacterium BB-12 in yogurt products by TaqMan-MGB fluorescent quantitative polymerase chain reaction(PCR).Methods The minor groove binder(MGB) probe with 16 S rRNA sequence of animal Bifidobacterium BB-12 was designed,and the overexpression vector of the target sequence was constructed by fluorescence quantitative PCR technology.The plasmid was extracted and the standard curve was made.Finally,commercial yoghurt samples were tested to quantify the number of BB-12 strains in yogurt.Results All the carrier standards showed significant fluorescence amplification,and the cycle threshold(Ct) was within the range of 16-20,however,DNA samples taken from other yogurt products containing bacteria and Escherichia coli as templates showed no fluorescent amplification signals.In terms of detection sensitivity,the mass concentrations at which the BB-12 carrier could be stably detected were as low as 0.0000584 ng/μL,and the average Ct value was 28.73,representing a detection sensitivity as low as 0.05 pg.The linearity of the drawn standard curve was good,and the amplification efficiency E was 0.39,with the determination coefficient R^(2)=0.9999.The commercial yogurt samples had good detection specificity,high sensitivity and accurate quantitative results.Conclusion The TaqMan-MGB probe fluorescence quantitative PCR method established in this study can realize the detection and quantification of BB-12 strain in yogurt.