基于反转录环介导等温扩增技术检测大肠杆菌O157

doi:10.13560/j.cnki.biotech.bull.1985.2017-0932

摘要/Abstract

摘要： 建立反转录环介导等温扩增（Reverse transcriptase loop-mediated isothermal amplification RT-LAMP）方法特异性检测大肠杆菌O157。针对大肠杆菌O157的特异性保守rfbE基因设计多组引物，通过对引物筛选和反应条件优化，建立了检测大肠杆菌O157的实时荧光RT-LAMP方法。利用大肠杆菌O157及其人工污染脱脂乳样品，研究了方法的特异性和灵敏度，并与rRT-PCR（Real-time fluorescent quantitative reverse transcription polymerase chain reaction）方法的灵敏度进行比较。结果显示，等温65℃条件下，30 min内能完成RT-LAMP扩增反应。所建立的实时荧光RT-LAMP方法特异性强，除了2株大肠杆菌O157以外其余菌株均未产生特异性扩增反应。同时该方法灵敏度高，对人工污染脱脂乳样品检测灵敏度能达到20 CFU/g，比rRT-PCR方法高10倍。建立的实时荧光RT-LAMP方法将为大肠杆菌O157现场快速检测提供有力手段。

关键词: 反转录-环介导等温扩增, 大肠杆菌O157, 检测, 脱脂乳

Abstract: A reverse transcriptase loop-mediated isothermal amplification（RT-LAMP）method was developed to specifically detect Escherichia coli O157. A number of primers were designed for specific conservative rfbE gene in E. coli O157. A real-time fluorescent RT-LAMP for detecting E. coli O157 was established by optimizing primer selection and reaction conditions. The specificity and sensitivity of the method were verified by using E. coli O157 and its artificial contaminated skim milk samples，and compared with the sensitivity of rRT-PCR（real-time fluorescent quantitative reverse transcription polymerase chain）. The RT-LAMP reaction was completed within 30 min under the condition of isothermal temperature of 65℃. RT-LAMP was highly specific since there was no specific amplification except two strains of E. coli O157. In the detection of artificial contaminated skim milk samples，the sensitivity reached 20 CFU/g，10 times of that by the rRT-PCR，meaning this method was highly sensitive. The established real-time fluorescent RT-LAMP will provide a powerful means for rapid detection of E. coli O157 on site.

Key words: reverse transcriptase loop-mediated isothermal amplification, Escherichia coli O157, detection, skim milk

梁玉林, 刘秀 ,周鹏飞 ,周振森 ,尹建军. 基于反转录环介导等温扩增技术检测大肠杆菌O157[J]. 生物技术通报, 2018, 34(6): 59-65.

LIANG Yu-lin, LIU Xiu ,ZHOU Peng-fei ,ZHOU Zhen-sen, YIN Jian-jun. Detection of Escherichia coli O157 by Reverse Transcriptase Loop-Mediated Isothermal Amplification[J]. Biotechnology Bulletin, 2018, 34(6): 59-65.

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