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

Abstract

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.

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR

“…In particular, the polymerase chain reaction (PCR) has been used successfully for the detection of Escherichia coli O157:H7, Listeria monocytogenes and Bacillus spp. in milk and other dairy products (Lantz et al 1994a;Candrian 1995;Dickinson et al 1995;Fratamico et al 1995;Herman et al 1995;Tsen et al 1996;Herman et al 1997). Many food components, however, are inhibitory to PCR, by either interfering with cell lysis (i.e., DNA extraction), contributing to nucleic acid degradation or inhibiting Taq DNA polymerase (Rossen et al 1992;Herman and De Ridder 1993;Powell et al 1994;Bickley et al 1996;Wilson 1997).…”

The application of PCR in the detection of mycotoxigenic fungi in foods

“…Analysis of specific nucleic acids in these products allows the determination of the presence or absence of certain ingredients or contaminants as well as the identification of specific characteristics of single components (7,32,35). DNA-based detection systems for genetically modified foods (17,21,22,24,25,31,53) and food-borne pathogens (1,2,21,27,51,55), including mycotoxin-producing fungi (6,8,9,18,42,47), have been developed recently. Furthermore, the detection of plant and animal species in the final food products has been shown to be feasible with DNA-based methods (33,34).…”

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

“…However, it was found that although the novel PCR primers for SLTI and SLTII genes detection have melting temperatures similar to those of the LTI and STII genes specific primers reported previously (Tsen et al 1996) i.e. 60°C vs 62°C, these primers could not be combined into a multiplex system for the simultaneous detection of the SLTI, SLTII, LTI and STII genes of E. coli cells.…”

“…Also, it should be mentioned that although a multiplex system that detects all types of ETEC (including STI, STII, LTI, and LTII E. coli) and STEC (including SLTI and SLTII E. coli) may be more useful, the simultaneous detection of any four, or more than four, genes has not been reported. Although we tried to combine the STI gene specific primers reported by us (Tsen et al 1996) or by Schultsz et al (1994) into out multiplex PCR system, the attempt was unsuccessful.…”

“…One of the considerations when designing the novel PCR primers for the SLTI and SLTII gene was that the annealing temperatures of the primers to be combined into a multiplex PCR system may be better if they were the same ; it may be easier to combine primers with similar annealing temperatures. However, it was found that although the novel PCR primers for SLTI and SLTII genes detection have melting temperatures similar to those of the LTI and STII genes specific primers reported previously (Tsen et al 1996) i.e. 60°C vs 62°C, these primers could not be combined into a multiplex system for the simultaneous detection of the SLTI, SLTII, LTI and STII genes of E. coli cells.…”