Rapid detection of E. coli causing diarrheal disease in human by using LAMP (Loop-mediated isothermal amplification) method

Abstract

Nowadays, many techniques are used for detecting pathogenic agents, including both traditional methods and modern molecular techniques. However, pathogenic Escherichia coli are not distinguishable from other common strains due to their appearance on culture plates or by the results of the usual biochemical tests. In this study, a new method called LAMP (Loop-mediated isothermal amplification) was applied to amplify DNA with high specificity and sensitivity at the detection limit of 15 pg, which was over 100 times higher than that of the PCR method. Four specific primers were designed to amplify the eae gene frogment - one of the toxic genes of E. coli with the length of 288 bp. Calcein (C30H26N2O13) is a fluorescent metal indicator for Magnesium detection. In this case, Calcein was initially quenched by manganese, then it emitted a yellowish-green fluorescence. In eae LAMP reaction, a large amount of DNA was synthesized, yielding a large pyrophosphate ion by-product. LAMP-generated pyrophosphate preferentially precipitated manganese, resulting in a complex and visible calcein-magnesium as bright green fluorescence. So a positive reaction was observed by a colour change from orange to green with the naked eyes after completion at 60°C for at least 30 min or under UV light detection. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method reveals a great potential in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing etc.