Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

Abstract

Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with 32P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae 01, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae 01, V. cholerae non-Ol (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli

“…c CFA (colonization factor antigen)/I, CFA/I pili [1]; type 1, type 1 pili; P, P pili. d EAST (enteroaggregative E. coli heat‐stable toxin 1) probe, see text; F1845 probe, 0.45‐kb Pst I fragment of pSLM852 which originated in the daaC gene on the chromosomal F1845 locus [15]; EAggEC probe, 0.8‐kb Pst I‐ Eco RI fragment of pCVD432 which originated in an adherence‐mediating plasmid of EAggEC [16]; LT (heat‐labile enterotoxin) probe, 0.85‐kb Hin cII fragment of pEW299 [13]; ST (heat‐stable enterotoxin) probe, 0.215‐kb Pst I‐ Bam HI fragment of pDAS100 [17]; EAF (EPEC adherence factor) probe, 1‐kb Bam HI‐ Sal I fragment of pMAR22 [18]; eaeA , EPEC attaching and effacing probe [19]; 17‐kb probe, 17‐kb Eco RI fragment of pRM17 [20]. e EAggEC with an EPEC serotype.…”

Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene

“…The technique was essentially as described for the colony immunoblotting except that the contact period was reduced to 5 min. Bacterial lysis, DNA denaturation and hybridization using the CFA/I-PI-P2 probe were performed as described previously (32,33).…”

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

“…Therefore, identification of the characteristic virulence genes is an obvious choice for DEC diagnostics. Since the discovery of the virulence genes, DNA hybridisation has been a successful method for the detection of DEC [5][6][7][8][9][10][11], but PCR has become the preferred diagnostic tool, because of the shorter time required for analysis and the simpler and less expensive experimental procedure. PCR analyses for identification of DEC groups have been designed that detect one or a few genes per reaction [10,[12][13][14][15][16][17][18][19][20][21][22][23][24].…”