Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens.
A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10(2) bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans. The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.
Fach
P
P
Centre National d'Etudes Vétérinaires et Alimentaires, Laboratoire Central d'Hygiène Alimentaire, Paris, France.
Guillou
J P
JP
D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005243:Feces / Q000382:microbiology; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D010738:Type C Phospholipases / Q000235:genetics*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D004269:DNA, Bacterial; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens