Open AccessTaguchi optimization of duplex PCR for simultaneous identification of S taphylococcus aureus and C lostridium perfringens alpha toxins
Author(s) -
Ramakrishna Uppalapati S.,
Kingston Joseph J.,
Harishchandra Sripathi Murali,
Batra Harsh V.
Publication year - 2013
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12070
Subject(s) - clostridium perfringens , staphylococcus aureus , microbiology and biotechnology , biology , polymerase chain reaction , clostridium , clostridiaceae , bacteria , toxin , gene , genetics
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 (274 bp) and cpaf/r to amplify clostridial alpha toxin gene cpa (398 bp) along with a competitive internal amplification control (608 bp), 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 10 pg of purified DNA or 10 3 CFU mL −1 of S. aureus and 100 pg of purified DNA or 10 4 CFU mL −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.