Detection by in Vitro Amplification of the Alpha-Toxin (Phospholipase C) Gene From Clostridium Perfringens

J Appl Bacteriol. 1993 Jan;74(1):61-6. doi: 10.1111/j.1365-2672.1993.tb02997.x.

Abstract

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.

MeSH terms

  • Bacterial Toxins / genetics*
  • Base Sequence
  • Calcium-Binding Proteins*
  • Clostridium perfringens / genetics*
  • Clostridium perfringens / isolation & purification
  • DNA, Bacterial / genetics
  • DNA-Directed DNA Polymerase
  • Feces / microbiology
  • Genes, Bacterial*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction*
  • Sensitivity and Specificity
  • Taq Polymerase
  • Type C Phospholipases / genetics*

Substances

  • Bacterial Toxins
  • Calcium-Binding Proteins
  • DNA, Bacterial
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • Type C Phospholipases
  • alpha toxin, Clostridium perfringens