Thin Film Biosensor for Rapid Detection of mecA from Methicillin-resistant Staphylococcus aureus

[摘要] The reported incidence of methicillin-resistant Staphylococcus aureus (MRSA) isolates in hospitals has increased from 2% in 1974 to 50% in 1997 (1). In addition, MRSA is emerging as a community-acquired pathogen. Resistance is most often mediated by the mecA gene, which encodes an altered penicillin-binding protein (PBP-2A) with low affinity for β-lactam antibiotics. Rapid identification of the mecA gene is important for implementation of appropriate antibiotic therapy.MRSA is identified by either culture or molecular methods. Routine culture methods require two sequential steps, one to isolate S. aureus and the second to determine antibiotic susceptibility (2). Recently, molecular methods, including PCR, branched DNA, and cycling probe assays, have been described that identify mecA sequences from individual S. aureus colonies (3)(4)(5)(6)(7). These methods are sensitive and specific, but they all require instrumentation to interpret the results.In this report, we describe a thin film biosensor for qualitative visual detection of mecA either directly from a single S. aureus colony or from a PCR amplification reaction. The technology allows direct visual detection of the interaction of target DNA sequences with complementary oligonucleotide probes immobilized to an optically coated silicon chip (8). The key assay steps for thin film formation are: (a) simultaneous hybridization of the target sequence to covalently attached surface capture probe and solution-phase biotinylated detector probes; (b) binding of anti-biotin antibody enzyme conjugate to the annealed detector probes; and (c) mass deposition through enzyme-catalyzed precipitation (Fig. 1A⇓ ). The thin film alters the interference pattern of light on the surface, producing a perceived color change. Composition of the optical layers is designed such that small …

[学科分类] 过敏症与临床免疫学