[Detection of enterohemorrhagic Escherichia coli O157:H7 by loop-mediated isothermal amplification].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
The Institute of Microbiology, Zhejiang Center for Disease Control and Prevention, Hangzhou, China.
Shui-rong
SR
Zhu
Yin
Y
Chen
Zhi-gang
ZG
Wang
Da-zhi
DZ
Jin
Qun-ying
QY
Lu
Ping-ping
PP
Yao
D004784:Environmental Monitoring / Q000379:methods*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity