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Development of Assays

Cepheid was one of the first companies to market an assay for methicillin-resistant Staphylococcus aureus (MRSA), based on its SmartCycler® real-time PCR platform.

Molecular detection of methicillin resistance in staphylococci is based on the detection of the mecA gene. However, since coagulase negative staphylococci (CNS) can also possess this gene, discrimination between CNS and MRSA must be achieved by the simultaneous detection of additional gene sequences specific for S. aureus. Cepheid's assay was a multiplex assay that did include targets for six variants of the mecA gene, as well as the S. aureus orfX gene. Despite this, independent investigators documented incidences of both false-positives and false-negatives.

The BD GeneOhm™ MRSA assay is another real time assay designed for the SmartCycler® platform. This assay employs molecular beacons for detection. The probe has a hairpin shape, with a fluorophore at one end, and a quencher at the other. In the absence of the target, the hairpin is closed and fluorescence is quenched. In the presence of the target, the hairpin opens when the beacon hybridizes to the target, resulting in the emission of fluorescence, which is measured during each cycle of amplification. Result availability is similar to the Cepheid assay. As with the Cepheid assay, independent investigators documented some incidence of both false positives and false negatives, but noted the advantage of rapid availability of screening results for surveillance purposes.