DEVELOPMENT OF A MULTIPLEX-PCR FOR DIRECT-DETECTION OF THE GENES FOR ENTEROTOXIN-B AND ENTEROTOXIN-C, AND TOXIC-SHOCK-SYNDROME TOXIN-1 IN STAPHYLOCOCCUS-AUREUS ISOLATES

As well as conventional methods such as immunodiffusion, ELISA, or agg lutination for the detection of toxin production in Staphylococcus aur eus, amplification techniques like PCR allow a very sensitive and spec ific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shoc k syndrome (TSS). For that reason an easy and quick test system for de termining the toxin production pattern of S. aureus isolates is desira ble so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex- PCR method was used that allowed single bacterial colonies grown on ag ar plates to be used directly in the PCR assay without preceding prepa ration. This procedure generated information concerning the presence o f seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-re sistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 o ther eubacterial isolates were tested initially with sets of single pr imer pairs followed by a combined multiplex-PCR. Results of this ampli fication technique were compared to a conventional and widely used met hod for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and spec ificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the cor responding toxin gene although the RPLA test did not show any detectab le toxin. The multiplex-PCR rapidly generated reliable information con cerning the toxin-producing capacity of staphylococcal strains and cou ld be easily integrated into a multiplex procedure described previousl y. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and m ecA genes.