实时荧光定量PCR法测定发酵乳中双歧杆菌

对实时荧光定量聚合酶链式反应(PCR)技术检测发酵乳中双歧杆菌的DNA提取方法、PCR扩增效率、标准曲线绘制进行探讨，通过比较分析碱式提取法、玻璃珠破碎法和酶解法3种提取方法对发酵乳中总DNA提取效率、4种不同参考菌株的PCR扩增效率以及单一菌株标准曲线与混合菌株标准曲线的计数结果，建立实时荧光定量PCR快速测定发酵乳制品中双歧杆菌数量方法。结果表明：酶解法提取发酵乳中总DNA效果最好，OD260/280比值基本接近1.80，且提取的质量浓度含量最高；不同菌株的PCR扩增效率不同，其中参考菌株1.2213的Ct值与其他3菌株的Ct值存在显著性差异；根据单一菌株绘制标准曲线与混合菌株绘制标准曲线计数结果无显著性差异，后者计数结果更客观、准确。采用酶解法获取样品中的菌体细胞，基于混合菌液绘制标准曲线，采用实时荧光定量PCR技术确定发酵乳中双歧杆菌种属数量，可快速、准确地测定发酵乳中双歧杆菌的数量。

Detection of Bifidobacteria in Fermented Dairy Products by Real-Time Fluorescent Quantitative PCR

In order to develop a rapid method for the detection of bifidobacteria in fermented milk products by real-time fluorescent quantitative PCR, the extraction efficiency of DNA in fermented milk products by different extraction methods, PCR amplification efficiency of four reference strains and enumeration of bifidobacteria according to different standard curves were compared. Results indicated that enzymatic hydrolysis with pronase E was the best extraction for total DNA with DNA OD260/280 ratio of 1.80. A significant difference between CT value of reference strain and the other strains was observed, and no significant difference between different standard curves was achieved. Therefore, real-time fluorescentn quantitative PCR technique is suitable for enumerating bifidobacteria that exist in commercial probiotic yoghurts.