Use of Novel Polymerase Chain Reaction Primers for the Specific Detection of Heat-Labile Toxin I, Heat-Stable Toxin I and II Enterotoxigenic Escherichia coli in Milk

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat-labile and/or heat-stable toxins (LT and ST) have been some of the most important microorganisms causing food- and waterborne diseases. Rapid and sensitive methods for the specific detection of enterotoxigenic E. coli are thus important. Although quite a few polymerase chain reaction (PCR) primers have been developed for the specific detection of ETEC genes coding for LT I, ST Ia, and ST Ib, only a few primers have been designed for the detection of ST II and ST Ia, together with ST Ib ETEC. By gene-sequence comparison and serial PCR assay studies, we were able to develop novel PCR primers specific for the detection of LT I and ST Ia as well as ST Ib and ST II enterotoxin-coding genes of E. coli cells. The DNA sequences of these PCR primers are different from those reported by other laboratories. Studies on the detection sensitivities of these PCR primers showed that when cell lysate rather than the total DNA obtained by the phenol-chloroform-isoamyl alcohol extraction method was used for PCR, a lower detection limit, i.e., 101 or 102 CFU target cells per assay could be obtained. When such a cell-lysis method was used for the PCR detection of ETEC cells in a variety of milk samples, such as whole, <2% fat, skim, and raw milk samples, it was found that if target cells in these milk samples were precultured in MacConkey broth for 8 h prior to cell lysis, as few as 100 cells per g of whole, <2% fat, or skim milk samples, and 102 cells per g of raw milk sample could be detected.