Synthesis and evaluation of a non-radioactive gene probe for the detection of C.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 plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc 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.putrifucum, 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 plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.
Schlapp
T
T
Institute of Hygiene and Infectious Diseases of Animals, Justus- Liebig-University, Giessen, Germany.
Blaha
I
I
Bauerfeind
R
R
Wieler
L H
LH
Schoepe
H
H
Weiss
R
R
Baljer
G
G
D001409:Bacillus cereus / Q000145:classification; D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002876:Chromosomes, Bacterial; D003013:Clostridium / Q000145:classification / Q000235:genetics; D003016:Clostridium perfringens / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000138:chemical synthesis*; D005798:Genes, Bacterial; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D013211:Staphylococcus aureus / Q000145:classification; D010738:Type C Phospholipases
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens