[1]俞正玉,张碧成,张强,等.鲜奶中肠出血性大肠杆菌O157∶H7的定量PCR检测[J].江苏农业学报,2015,(05):1173-1178.[doi:doi:10.3969/j.issn.1000-4440.2015.05.036]
　YU Zheng-yu,ZHANG Bi-cheng,ZHANG Qiang,et al.Development of a real-time PCR for detection of enterohemorrhagic Escherichia coli O157∶H7 in milk[J].,2015,(05):1173-1178.[doi:doi:10.3969/j.issn.1000-4440.2015.05.036]
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鲜奶中肠出血性大肠杆菌O157∶H7的定量PCR检测()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年05期
页码:
1173-1178
栏目:
加工贮藏·质量安全
出版日期:
2015-10-31

文章信息/Info

Title:
Development of a real-time PCR for detection of enterohemorrhagic Escherichia coli O157∶H7 in milk
作者:
俞正玉; 张碧成; 张强; 汪伟; 何孔旺; 倪艳秀; 温立斌; 李彬; 周萍; 张雪寒
（江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心，江苏南京210014）
Author(s):
YU Zheng-yu; ZHANG Bi-cheng; ZHANG Qiang; WANG Wei; HE Kong-wang; NI Yan-xiu; WEN Li-bin; LI Bin; ZHOU Ping; ZHANG Xue-han
(Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural  Ministry/National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China)
关键词:
肠出血性大肠杆菌O157∶H7; 荧光定量PCR; 鲜奶
Keywords:
enterohaemorrhagic Escherichia coli O157∶H7; real time fluorescence quantitative PCR; milk
分类号:
S851.34+7.502
DOI:
doi:10.3969/j.issn.1000-4440.2015.05.036
文献标志码:
A
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1 μl 1×101拷贝至1 μl 1×106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1 μl 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。
Abstract:
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 template to develop RT-PCR assay. The concentration of stantard plasmid exhibited a good linear relationship with Ct within the range of 1×101-1×106copies per microlitre by developed RT-PCR, with the correlation coefficient being 0.996 and the amplification 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 coefficients of variation within and among batches were lower than 2%, suggestive of good repeatability.

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备注/Memo

备注/Memo:
收稿日期：2015-04-05 基金项目：国家自然科学基金项目（31572503）；农业部948项目（2014-S17） 作者简介：俞正玉（1978-），男，江苏南京人，学士，助理研究员，从事人兽共患病研究。 通讯作者：张雪寒，（E-mail）liuxuehan1996@hotmail.com
更新日期/Last Update: 2015-10-31