Improved method for the routine identification of toxigenic Escherichia coli by DNA amplification of a conserved region of the heat-labile toxin A subunit

Abstract

This report describes a DNA amplification procedure for routine identification of heat-labile-toxin-producing Escherichia coli. Two oligonucleotide primers were used in a polymerase chain reaction procedure to amplify a highly conserved region of the A subunit of the heat-labile enterotoxin gene. Amplifications were done directly on E. coli colonies from plates when Salmonella, Shigella, or parasite infections were excluded as agents of the severe diarrhea in the patients. The conditions for the polymerase chain reaction method were empirically determined, and the procedure is inexpensive, sensitive, and specific. Positive results can be obtained over a wide variation in bacterial numbers, with no inhibition of Thermus aquaticus DNA polymerase. Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of this pathogen in clinical isolates. If greater sensitivity and specificity are required, hybridization with 32p_ or alkaline phosphatase-labeled oligonucleotide probes can be used. Our results suggest that heat-labile-toxin-producing E. coli is responsible for about 9% of nondiagnosed diarrhea cases in Tygerberg Hospital, Tygerberg, Republic of South Africa.

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

“…In order to develop a multiplex PCR system for the simultaneous detection of SLTI, SLTII, LTI and STII genes of E. coli cells, several other combinations of primers were tried. After several attempts, it was found that primers SLTI-5/SLTI-3 and SLTII-5/SLTII-3 could be combined with the LTI and STII genes specific primers reported by Victor et al (1991) and Lortie et al (1991) and that the detection specificity of this multiplex system is reliable. Under the PCR conditions described under Materials and Methods, all the cells tested could generate the expected PCR products, i.e.…”

“…In addition to the novel PCR primers designed by this laboratory for the detection of the SLTI gene (primer SLTI-5/SLTI-3) and SLTII gene (primer SLTII-5/SLTII-3), PCR primers specific for LTI (primer LT 5-1/LT 3-1) and STII gene (primer STII-FP/STII-RP) reported by Victor et al (1991) and Lortie et al (1991) were used. The sequences, locations within genes, Tm values, and the sizes of PCR products are shown in Table 2.…”

“…The cell lysate used for PCR was prepared by a method modified from Victor et al (1991). For PCR amplification of the SLTI and SLTII genes, each 0·5 ml Eppendorf tube contained 1 ml each of 10 mmol l −1 dATP, dTTP, dGTP and dCTP, 4 ml of 10×PCR buffer (100 mmol 1 −l Tris-HCl, pH 8·8, 500 mmol l −1 KCl, 15 mmol l −1 MgCl 2 , and 1% Triton X-100), 25 rmole each of the primers (SLTI-5/SLTI-3 or SLTII-5/SLTII-3), 10 ml of the target cells (10 6 cfu 10 ml −1 ) and sterilized H 2 O (to a final volume of 40 ml).…”

Designing of polymerase chain reaction primers for the detection of Salmonella enteritidis in foods and faecal samples

“…For the PCR assay, the cell lysate was used as source material and the method of Victor et al . (1991) was modified for cell lysate preparation.…”

Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction

“…However, it has been shown for other organisms (13,23) and for Mycobacterium spp. (6,19) that PCR amplification can be done successfully on crude sample preparations without prior DNA extraction.…”