Zhu Shui-rong, Chen Yin, Wang Zhi-gang, Jin Da-zhi, Lu Qun-ying, Yao Ping-ping
The Institute of Microbiology, Zhejiang Center for Disease Control and Prevention, Hangzhou, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2009 Sep;43(9):803-8.
To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories.
Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method.
The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection.
LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.
开发一种环介导等温扩增（LAMP）方法，用于病原体检测部门或小型实验室快速诊断大肠杆菌O157:H7。
分别针对大肠杆菌O157:H7的抗原编码基因rfbE、志贺样毒素stx2以及H7鞭毛抗原的fliC编码基因设计LAMP检测引物。优化LAMP反应条件和反应体系。对2株大肠杆菌O157:H7标准菌株、17株本地菌株和33株其他肠道杆菌进行分析，以评估LAMP的特异性和敏感性。LAMP反应结果还与常规PCR方法进行比较。
具有相应靶基因的O157扩增产物肉眼观察呈绿色，凝胶电泳呈梯状条带，而其他肠道杆菌产物肉眼观察仍为橙色，凝胶电泳无条带。LAMP检测结果与常规PCR方法一致。LAMP方法的反应时间仅为1.5小时，检测限为26 CFU/反应。此外，LAMP结果仅通过肉眼观察即可判定。
LAMP检测对大肠杆菌O157:H7的检测具有快速、特异、灵敏的特点。该方法不仅可减少对复杂设备的依赖，还可能成为在病原体检测部门、小型实验室、应急机动车辆或现场调查中更广泛应用的潜在方法。
