Taguchi optimization of duplex PCR for simultaneous identification ofStaphylococcus aureusandClostridium perfringensalpha toxins

Abstract

Staphylococcus aureus and Clostridium perfringens are two major bacteria that infect open wounds and delay the healing process. The rapid and progressive deterioration of soft tissue during S.aureus and C.perfringens coinfections is due to analogous necrotic alpha toxins produced by the two organisms. The aim of this study was to determine the alpha toxins of S.aureus and C.perfringens by duplex PCR. The PCR assay employed two sets of primers: hlaf/r to amplify staphylococcal alpha toxin gene hla (274bp) and cpaf/r to amplify clostridial alpha toxin gene cpa (398bp) along with a competitive internal amplification control (608bp), simultaneously. Optimization of the duplex PCR assay was achieved by a modified Taguchi method, an engineering optimization process, in a nine-tube combinatorial array. The detection level of the duplex PCR was found to be 10pg of purified DNA or 10(3) CFUmL(-1) of S.aureus and 100pg of purified DNA or 10(4) CFUmL(-1) of C.perfringens. Other bacteria routinely found in tissue infections were tested for cross-reactivity and the duplex PCR turned out to be highly specific. This duplex PCR assay provides a rapid, robust and reliable alternative to the existing conventional techniques in establishing the aetiology of S.aureus and C.perfringens in soft tissue infections.