SYNTHESIS AND EVALUATION OF A NONRADIOACTIVE GENE PROBE FOR THE DETECTION OF CLOSTRIDIUM-PERFRINGENS ALPHA-TOXIN

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned pie gene of C. perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C. perfringens were tested for pie. None of the strains failed in hybridization. Presence of pie was even demonstrated in C. perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp, tested, e.g. C. bifermentans, C. tertium, C. novyi, C. chauvoei, C. sporogenes, C. difficile, C. putrificum, C. sordellii, C. botulinum, C. septicum and C. histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the pie probe proved to be a much more sensitive and specific diagnostic tool for the detection of C. perfringens plc.