A sensitive method for the detection of enterotoxigenic Escherichia coli by the polymerase chain reaction using multiple primer pairs

Abstract

In this study, a polymerase chain reaction (PCR) method has been developed for the detection of enterotoxigenic Escherichia coli (ETEC). Three different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STIa and STIb) enterotoxins of ETEC. These primers amplified a 627, 240, or 169 base pair (bp) DNA fragment from LTh, STIa and STIb gene, respectively, of the reference ETEC strains. The addition of RNase A (10 micrograms/ml) to the PCR reaction solution diminished nonspecific amplification of DNA fragments other than the enterotoxin genes. Five types of ETEC strains corresponding to the LTh, STIa, STIb, LTh-STIa, or LTh-STIb genotypes were distinguished by a single procedure of PCR using the mixture of the three sets of primers. PCR, hybridization, and conventional methods were subjected to one hundred stool specimens from diarrheal patients. It was found that PCR was the most sensitive method among them. These results suggested that PCR with triple primer pairs would be useful for the laboratory diagnosis of ETEC in the stool specimens.