Detection of toxigenic Escherichia coli using biotin-labelled DNA probes following enzymatic amplification of the heat labile toxin gene

Abstract

Several types of DNA probes labelled with biotin were compared for their sensitivity to detect the heat labile toxin (LT) gene in toxigenic Escherichia coli. In addition, a procedure was developed for enzymatically amplifying LT gene sequences in toxigenic E. coli. Probes were labelled with biotinylated nucleotides by either nick translation; 3′ tailing; primer extension of probe DNA cloned into bacteriophage M13; sandwich hybridization; or oligolabelling of isolated DNA fragments. A single stranded probe consisting of a DNA fragment from the LT gene cloned into the bacteriophage M13mp18 and detected by hybridization to oligolabelled biotinylated M13mp18 RF DNA in a sandwich hybridization was able to detect as little as 10 pg of toxin gene DNA. Cloned LT gene DNA was serially diluted and amplified enzymatically using synthetic oligonucleotide primers. Amplified DNA was detected using biotin-labelled M13-based probes. As little as 1 fg of LT DNA could be amplified to detectable levels by this method. Experiments with LT + bacteria resulted in the detection of as few as 1000 bacteria. The combination of enzymatic amplification coupled with M13-based DNA probes provides a highly sensitive tool for detecting pathogenic microorganisms.