大肠杆菌O157︰H7特异基因的荧光定量PCR检测

doi:10.7506/spkx1002-6630-201112061

食品科学 ›› 2011, Vol. 32 ›› Issue (12): 278-282.doi: 10.7506/spkx1002-6630-201112061

大肠杆菌O157︰H7特异基因的荧光定量PCR检测

胡慧，陈雅君，段志刚，孟振北，彭新然，张龙现，崔保安，王亚宾

1. 河南农业大学牧医工程学院
2.漯河出入境检验检疫局

出版日期:2011-06-25 发布日期:2011-06-10
基金资助:
河南省重大公益科研项目(81100912300)；“十一五”国家科技支撑计划项目(2007BAQ01047)；漯河市科技计划项目(081203)

Real-time PCR Detection of Specific Gene in Escherichia coli O157:H7

HU Hui1，CHEN Ya-jun1，DUAN Zhi-gang1，MENG Zhen-bei2，PENG Xin-ran2，ZHANG Long-xian1， CUI Bao-an1，WANG Ya-bin1,*

1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China； 2. Luohe Entry-exit Inspection and Quarantine Bureau, Luohe 450046, China

Online:2011-06-25 Published:2011-06-10

摘要/Abstract

摘要： 为建立快速、特异的检测大肠杆菌O157:H7的实时荧光定量聚合酶链式反应(real time polymerase chain reaction，RT-PCR)方法，针对大肠杆菌O157:H7的特异基因rfbE设计一对特异引物，建立SYBR GreenⅠ实时定量PCR检测方法，并进行灵敏度、重复性和特异性实验，同时与常规PCR方法进行比较。结果显示所建立的SYBR GreenⅠ实时定量PCR方法可以快速、特异地检测出大肠杆菌O157:H7，细菌纯培养物中其灵敏度可达2×101CFU/mL，临床模拟污染肉样中能最低能检测到1×102CFU/mL的大肠杆菌O157:H7。与常规PCR方法相比，SYBR GreenⅠ实时定量 PCR方法对临床样品中大肠杆菌O157:H7的检出率大大提高。本研究建立的SYBR GreenⅠ荧光定量 PCR技术能快速准确、特异、敏感地检测大肠杆菌O157:H7。

关键词: 大肠杆菌O157︰H7, rfbE基因, SYBR Green I, real-time PCR

Abstract: A real-time PCR method was developed for the rapid and specific detection of Escherichia coli O157:H7. A pair of primers was designed according to the conserved sequence of rfbE gene in Escherichia coli O157:H7. The SYBR Green I real-time PCR method for detecting Escherichia coli O157:H7 was established. The sensitivity and specificity of this method was analyzed through the comparison with traditional PCR methods. The results indicated that the developed SYBR Green I real-time PCR method had the characteristics of excellent specificity, sensitivity and repeatability. The sensitivity of this developed method was 2 × 101 CFU/mL in pure cultures and 1 × 102 CFU/mL in artificially contaminated meat samples. In addition, the established method was also used for the detection of clinical samples. The results showed that the detection rate of real-time PCR for Escherichia coli O157:H7 was significantly increased when compared with traditional PCR. Therefore, the established real-time PCR method is a rapid, specific and sensitive method for the detection of Escherichia coli O157:H7.

Key words: Escherichia coli O157:H7, rfbE gene, SYBR Green I, real-time PCR

中图分类号:

R378.21

胡慧，陈雅君，段志刚，孟振北，彭新然，张龙现，崔保安，王亚宾. 大肠杆菌O157︰H7特异基因的荧光定量PCR检测[J]. 食品科学, 2011, 32(12): 278-282.

HU Hui，CHEN Ya-jun，DUAN Zhi-gang，MENG Zhen-bei，PENG Xin-ran，ZHANG Long-xian，CUI Bao-an，WANG Ya-bin. Real-time PCR Detection of Specific Gene in Escherichia coli O157:H7[J]. FOOD SCIENCE, 2011, 32(12): 278-282.

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大肠杆菌O157︰H7特异基因的荧光定量PCR检测

Real-time PCR Detection of Specific Gene in Escherichia coli O157:H7

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