利用环介导等温扩增技术快速检测肠出血性大肠杆菌及其毒素的方法建立和评价  

Development and evaluation of loop-mediated isothermal amplification assay for the rapid detection of Escherichia coli and its microbial toxin

作　　者：钟玉葵 邓丽丝 邓秋连 钟华敏 骆明勇[3] 周珍文 颜慕霞 谢永强 Zhong Yukui;Deng Lisi;Deng Qiulian;Zhong Huamin;Luo Mingyong;Zhou Zhenwen;Yan Muxia;Xie Yongqiang(Department of Clinical Laboratory, Guangzhou Women and Children＇s Medical Ceter, Guangzhou 510120 , China;Department of Emergency, Guan- gzhou Huadu District People＇s Hospital, Guangzhou 510800, Chin;Medical Genetic Centre, Guangdong Women and Children Hospital, Guangzhou 511400, Chin)

机构地区：[1]广州市妇女儿童医疗中心检验科,510120 [2]广州市花都区人民医院急诊科,510800 [3]广东省妇幼保健院医学遗传中心,广州511400

出　　处：《中国医师杂志》2018年第6期826-831,共6页Journal of Chinese Physician

基　　金：广东省省级科技计划项目(2015A030401007)~~

摘　　要：目的针对编码大肠杆菌志贺毒素由位于STEC染色体上的前噬菌体携带的stx基因和O_(157)抗原编码基因rfbe设计特异性引物,并对反应体系和反应条件进行优化,建立一种针对肠出血性大肠杆菌及其毒素的环介导等温扩增(LAMP)反应技术检测法。方法通过优化LAMP反应的各影响因素,确定LAMP反应体系和反应条件并利用已优化的LAMP体系进行相关检测。结果确定LAMP反应体系为:内引物(FIP、BIP)与外引物(F3、B3)的比例为8:1,Mg^(2+)浓度10 mmol/L,dNTPs浓度1. 2 mmol/L,甜菜碱浓度0. 4 mol/L,Bst DNA聚合酶的添加量为1μl。LAMP反应时间为60 min,反应温度为60。以PCR法作为对照,LAMP检测的敏感度为5×10~1 CFU/mL,PCR的敏感度为5×10~4 CFU/mL。同时,研究应用所建立的LAMP快速检测法对部分革兰阳性和阴性菌及102株O_(157)大肠杆菌进行快速检测。结果显示,检测特异度达100%,O_(157)大肠杆菌rfbe检出率为100%(102/102),stx1检出率为95. 2%(59/62),stx2检出率为92. 9%(52/56)。结论成功建立了一种可应用于肠出血性大肠杆菌及其毒素的敏感、特异的LAMP检测方法。Objective To establish and optimize a loop-mediated isothermal amplification （LAMP） method for the rapid detection of Escherichia coli and its microbial toxin. Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction, and the optimized LAMP system was used for the detection. Results Primers targeting shiga toxin （stx） gene and O157 antigen gene rfbe were designed. The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs, 10 mmol/L MgSO4, 0. 4 mol/L betaine, 1 μl 10 × Bst DNA polymerase Buffer, 8 U Bst DNA polymerase fragment, 2 pl DNA template, and the ratio of inner-primer （FIP and BIP） and outerprimer （F3 and B3） were 8： 1. Time and temperature for LAMP was 60 min, 60℃. The sensitivity was 103 times higher than polymerase chain reaction （PCR）, reached 5 × 10 1 CFU/ml. When LAMP was applied to 19 reference strains, 102 EHEC strains, the specification was 100% while identification rate of rfbe, stxl and stx2 gene reached 100%, 95.2%, 92. 9%. Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.

关 键 词：肠出血性大肠杆菌/微生物学 核酸扩增技术 志贺毒素/遗传学 细菌蛋白质类/遗传学 

分 类 号：R446.5[医药卫生—诊断学]