COLORIMETRIC DETECTION OF HEAT-LABILE TOXIN-ENCODING GENE OF ENTEROTOXIGENIC ESCHERICHIA-COLI BY PCR

In the developing world, enterotoxigenic Escherichia coli (ETEC) strai ns which produce enterotoxins are a significant cause of morbidity and mortality. Heat-labile (LT) toxin PCR detection methods have been des cribed, but they have limited applications in a routine laboratory set ting. A colorimetric DNA method for the rapid amplification and detect ion of the LT toxin gene in ETEC strains is described. Target amplific ation together with colorimetric detection would overcome many of the limitations of conventional PCR. This paper describes a colorimetric P CR detection method specific for LT-gene-encoding ETEC strains. DNA wa s extracted from two representative colonies from each bacterial isola te and amplified by PCR. Digoxigenin was incorporated into the amplifi cation product, permitting a one-step direct detection using anti-digo xigenin alkaline phosphatase-conjugated antibody. This technique was a pplied to the investigation of 70 E. coli isolates derived from clinic al fecal samples obtained from an Irish population. Eleven percent of the samples were LT positive, confirming the applicability of this met hod. All LT-positive ETEC strains (controls and clinical isolates) wer e detected, and no false-positive results occurred.