Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene

Abstract

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies.

FIG. 1

Agarose gel electrophoresis patterns showing PCR-amplified products in multiplex PCR for the staphylococcal enterotoxin genes. Lanes 1, DNA molecular weight marker λ HindIII; 2, sea; 3, seb; 4, sec; 5, sed; 6, see; 7, multiplex PCR with all enterotoxin genes simultaneously (sea to see); 8, artificial arrangement of the amplification fragments of sea to see; 9, DNA molecular weight marker λ BstE II. Sizes are marked in base pairs on the left and right.

FIG. 2

Agarose gel electrophoresis patterns showing PCR-amplified products in multiplex PCR for the staphylococcal ET genes (eta and etb) and TSST-1 gene (tst). Lanes 1: DNA molecular weight marker λ HindIII; 2, eta; 3, etb; 4, tst; 5, multiplex PCR with both ET genes and the TSST-1 gene simultaneously; 8, artificial arrangement of the amplification fragments of eta, etb, and tst; 9, DNA molecular weight marker λ BstE II. Sizes are marked in base pairs on the left and right.