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. LAMPgenerated pyrophosphate preferentially precipitated
manganese, resulting in a complex and visible calceinmagnesium 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.