I . 1998. The primary sequences of the V 3 and V 6 regions of the 16S rRNA gene of pathogenic and non-pathogenic strains of Escherichia coli were determined and compared with those obtained for a number of reference strains which belong to the family Enterobacteriaceae. Three oligonucleotide primers 16E1, 16E2 and 16E3 were designed and used in the polymerase chain reaction to identify specifically all E. coli isolates. When 16E1, 16E2 and 16E3 were used as primers for the identification of E. coli cells present in tap, underground and pond waters, as low as 1 cfu 100 ml −1 of water could be detected if an 8 h pre-culture step was performed prior to the PCR reaction.

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT 1-and ST 11-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 10(0) cells per ml of the sample could be detected. Without the preculture step, 10(4) CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR system can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

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.