Detection byin vitroamplification of the alpha‐toxin (phospholipase C) gene fromClostridium perfringens

摘要

A polymerase chain reaction (PCR) with thermostable DNA polymerase fromThermus aquaticusis described for the specific amplification of the phospholipase C (alpha‐toxin) gene ofClostridium perfringens.A set of primers selected for their high specificity could detectCl. perfringensin stools with a detection limit of approximately 5 x 102bacteria, 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 methodCl. perfringensalpha‐toxin gene could be detected within 2 h. The PCR method detected alpha‐toxin positiveCl. perfringensbut did not react with phospholipase C‐producingBacillus cereus, Pseudomonas aeruginosa, Cl. sordelliiandCl. 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 pathogenCl. perfringensas it may be used directly on stoo