Sensitivity of detection of enterotoxigenic Escherichia coli from stool sample by DNA probe

An attempt has been made to detect the minimum counts of enterotoxigenic Escherichia coli (ETEC) in stool sample under simulated clinical condition. Thermostable (ST-la) enterotoxin-producing ETEC culture was mixed with stool sample and normal saline, centrifuged, then the supernatant was further diluted with saline and different volumes were spotted on nitrocellulose paper. Hybridization with 32P labelled pDAS-101 DNA and viable count of original culture on MacConkey agar plates with ampicillin revealed that minimum 8 cells of ETEC (ST) could be detected. The method of labelling used was sequential harnessing of the catalytic and synthetic activity of the large Klenow fragment of DNA polymerase-I. Linearizing of the DNA was dispensed with as the nicked circular DNA was excised with the gel and used for labelling directly.