摘要
为建立鲜奶中肠出血性大肠杆菌(Enterohaemorrhagic E.coli,EHEC)O157∶H7的特异性检测方法,以EHEC O157∶H7独有的遗传标志性基因Z0372为靶序列设计引物和探针,以梯度稀释的含有Z0372基因的重组质粒作为标准品进行Taqman荧光定量PCR反应,分析该方法的敏感性、特异性,并检测鲜奶样品以验证Taqman荧光定量PCR实用性和可靠性。结果表明,建立的定量PCR方法在1μl 1×101拷贝至1μl 1×106拷贝之间具有良好的线性关系,相关系数达到0.996,扩增效率112%。该方法的检测灵敏度为1μl 70拷贝,敏感性较高,而且特异性良好,与其他血清型大肠杆菌、沙门氏杆菌和志贺氏菌等易混淆菌株核酸之间没有交叉反应。批间和批内检测的变异系数(RSD)低于2%,表明该方法重复性良好。
To develop a real-time PCR ( RT-PCR ) technique for detection of enterohaemorrhagic Escherichia coli ( EHEC) O157 ∶ H7 in milk, primers and probes were designed according to the unique Z0372 gene sequence, a genetic marker of EHEC O157 ∶ H7, and the serially diluted recombinant plasmid pMD18-T-Z0372 was used as a standard tem-plate to develop RT-PCR assay. The concentration of stantard plasmid exhibited a good linear relationship with Ct within the range of 1×101-1×106copies per microlitre by developed RT-PCR, with the correlation coefficient being 0. 996 and the am-plification efficiency being 112%. The RT-PCR assay showed a detection limit as low as 70 copies per microlitre and no cross reaction with other E. coli serotypes, Salmonella and Shigella, indicative of good sensitivity and speificity. The coeffi-cients of variation within and among batches were lower than 2%, suggestive of good repeatability.
出处
《江苏农业学报》
CSCD
北大核心
2015年第5期1173-1178,共6页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31572503)
农业部948项目(2014-S17)
关键词
肠出血性大肠杆菌O157∶H7
荧光定量PCR
鲜奶
enterohaemorrhagic Escherichia coli O157∶ H7
real time fluorescence quantitative PCR
milk
