Detection of Escherichia coli Producing Heat-Stable Enterotoxin II by Using ELISA and PCR.

Abstract

Because of the lack of the convenient assay to detect the heat-stable enterotoxin II of Escherichia coli (STII), STII produced from E. coli in clinical isolates has not been inspected in regular clinical examinations. And it has not been examined whether these isolates possess a gene for STII, too, as a colony hybridization test which is the conventional method to detect strains possessing the specified gene of bacteria is a troublesome work for inspectors in regular bacteriological examination laboratory. In this study, we developed two methods to detect STII and its gene. One was the enzyme-linked immunosorbent assay (ELISA) to detect STII and the other was the polymerase chain reaction (PCR) to detect the gene for STII. Using these methods, we examined STII and its gene from 120 strains isolated from stools of porcines with diarrhea in Brazil. At first, a colony hybridization test employing a DNA fragment encoding the STII gene was performed to find strains possessing the STII gene. Twenty-three strains out of 120 porcine strains were found to possess STII genes. In ELISA, samples from these 23 strains which were positive in the colony hybridization test showed positive reaction and those from other strains which were negative in the colony hybridization test showed negative reaction. To apply PCR for the detection of strains possessing the STII gene, we designed the primer set to detect a 380-base pair (bp) fragment. The 380-bp fragments were enzymaticaly amplified from DNAs of these 23 strains which were positive in the two tests described above but not from those of other strains which were negative in these tests. These results showed that ELISA and PCR developed in this study are specific for STII and that these developed methods are useful for the screening of large number of clinical strains.