荧光定量PCR方法快速检测原料乳中的大肠杆菌  

SYBR GreenⅠ fluorescence quantitative PCR detection of Escherichia coli in raw milk

作　　者：周微[1] 张伟钦[1] 付宇[1] 贡汉生[1] 孟祥晨[1] 

机构地区：[1]东北农业大学乳品科学教育部重点实验室/食品学院,哈尔滨150030

出　　处：《中国乳品工业》2009年第11期39-42,共4页China Dairy Industry

基　　金："十一五"国家科技支撑计划(2006BAD04A08)资助项目

摘　　要：利用SYBR GreenⅠ荧光定量PCR技术建立一种快速检测原料乳中大肠杆菌数量的方法。根据大肠杆菌的ITS保守序列设计特异性引物,应用常规PCR方法确定引物特异性,然后优化SYBR GreenⅠ的添加量和Mg2+的额外添加量,确定对原料乳中大肠杆菌的最低检测限。结果表明,引物具有很强的特异性,最终确定的SYBR GreenⅠ(20×)的添加量为0.75μL,Mg2+的额外添加量为0.875mmol/L;在不增菌的前提下,对原料乳中大肠杆菌的最低检测限为1.7×103mL-1。检测的10份样品中有四份样品的大肠杆菌数超过103mL-1,与平板菌落计数结果无显著差异。该方法特异性强,灵敏度高,操作简便快捷,整个检测过程仅需3h,具有较大的推广及应用价值。To establish a rapid detection method of E.coli with SYBR GreenⅠfluorescence quantitative PCR in raw milk. A pair of amplify primers were designed to amplify the internal transcribed spacer region （ITS）, and then E.coli standard bacterium strain was applied for template. The primer specificity were determined with conventional PCR, then quantity of SYBR Green Ⅰand Mg^2＋ were optimized. The standard curve was established and the sensitivity test was performed. The results showed that the primers were highly conservative and specific.The optimized SYBR Green Ⅰ （20x）addition in this research is 0.75 μL and Mg^2＋ concentration is 0.875 mmol/L. The quantitative detection limit of the method was 1.7×10^3 cfu/mL in raw milk without increasing bacteria step.During 10 raw milk samples, the E.coli concentration beyond 10^3 mL^-1 were found in 4 samples, and have no significant differences compared with plate culture count. The method is specific, sensitive and rapid. The whole detection can be finished in 3 h.

关 键 词：大肠杆菌 原料乳 荧光定量PCR 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]