Multiplex PCR for the Rapid Simultaneous Speciation and Detection of Methicillin-Resistance and Gene

The use of polymerase chain reaction (PCR) to rapidly amplify target DNA molecules has evolved a place within diagnostic microbiology allowing the sensitive and specific identification of microorganisms concurrent with the detection of specific genes involved in resistance or virulence. This is particularly helpful when dealing with problem pathogens such as Staphylococcus aureus , where the rapid diagnosis and detection of methicillin-resistance can assist in the fast implementation of appropriate treatment and infection control measures (1 -4 ). In addition, the rapid assessment of toxin production can ascertain whether toxin-related symptoms need to be considered (5 ). The protocol described here enables the simultaneous differentiation between S. aureus (and coagulase negative staphylococci) from other eubacterial organisms. Concomitant with species identification this protocol allows for the detection of specific staphylococcal resistance genes, such as mec A, encoding methicillin resistance, or staphylococcal virulence genes, such as seb, sec1 and tst , encoding enterotoxin B, enterotoxin C, and toxic shock syndrome toxin-1, respectively. In S. aureus the detection of methicillin-resistance using agar diffusion or broth micro-dilution techniques, or toxin production using immunodiffusion, agglutination, or ELISA is often difficult. This is because the mec A gene and the genes involved in toxin production may be poorly expressed and influenced significantly by culture conditions (5 -10 ). Other methods, in particular various hybridization techniques have been used as diagnostic tools. However,