Use of biotinylated DNA probes in colony hybridization

Detection of enterotoxigenic Escherichia coli by dot blot hybridization with biotinylated DNA probes.

A dot blot hybridization test was developed for the detection of enterotoxigenic E. coli without the use of radioisotopes. Three biotin-labeled DNA (Bio-DNA) probes corresponding to structural genes specifying heat-labile and heat-stable enterotoxins of porcine and human origin were prepared by random priming; label incorporation was significantly higher than that obtained from the use of nick ...

Labeling of in situ hybridization DNA probes

Identification of enterotoxigenic Escherichia coli by colony hybridization with nonradioactive digoxigenin-labeled DNA probes.

Enterotoxigenic Escherichia coli (ETEC) strains were readily identified in pure and mixed cultures with nonradioactive, digoxigenin-labeled DNA probes coding for heat-labile (LTI) and heat-stable (STaI, STaII, and STb) enterotoxins. Digoxigenin-labeled ETEC fragments were more sensitive than and exhibited less nonspecific background contamination than biotinylated ETEC probes.

Increasing the specificity of colony hybridization when using heterologous probes.

Goat anti-mouse immunoglobulin horseradish peroxidase (HRP) conjugate was diluted in PBS containing 10% BSA. This conjugate has been successfully used from various manufacturers; however, specific lots need to be titrated for optimal performance. One particular preparation of HRPconjugated goat anti-mouse IgG, IgM and IgA (Catalog No. A 0412; Sigma Chemical, St. Louis, MO, USA) was diluted at 1...

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain t...