Evaluation of an isothermal signal amplification method for rapid detection of methicillin-resistant Staphylococcus aureus from patient-screening swabs

Abstract

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). The assay targeted the coa (coagulase) and mecA genes, thereby simultaneously identifying S. aureus and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 x 10(5) and 10(6) CFU/assay, equivalent to 4 x 10(6) to 2 x 10(7) CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a mecA-femB PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10(2) to 10(5) CFU/assay (below the CytAMP detection limit of 2 x 10(5) CFU/assay), and the sixth contained 10(6) CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.

FIG. 1.

Initial stages in the CytAMP assay format. (A) The short extension probe forms a 3WJ in the presence of the target sequence and the template probe. (B) The template probe contains a single-stranded T7 RNA polymerase promoter sequence, which is converted to a functional double-stranded promoter by Bst DNA polymerase extension from the extension probe. (C) Multiple copies of an RNA signal sequence are transcribed by T7 RNA polymerase from the template probe. This RNA sequence forms a substrate for additional rounds of extension and transcription in a second reaction that generates an unrelated signal sequence (12, 24). All reactions occur concomitantly in a single microcentrifuge tube.

FIG. 2.

Signal/noise ratios for mecA obtained in a typical experiment with five duplicate assays and decreasing numbers of MRSA in each sample. Error bars, standard errors. The positive cutoff value of 2.0 was that recommended for the prototype assay by the manufacturer.