Real-time nucleic acid sequence-based amplification assay for rapid detection and quantification of agr functionality...

Staphylococcus aureus infections are a significant cause of morbidity and mortality in health care settings. S. aureus clinical isolates vary in the function of the accessory gene regulator (agr), which governs the expression of virulence determinants, including surface and exoproteins, while agr activity has been correlated with patient outcome and treatment efficiency. Here we describe a duplex real-time nucleic acid sequence-based amplification (NASBA) detection and quantification platform for rapid determination of agr functionality in clinical isolates. Using the effector of agr response, RNAIII, as the assay target, and expression of the gyrase gene (gyrB) as a normalizer, we were able to accurately discriminate agr functionality in a single reaction. Time to positivity (TTP) ratios between gyrB and RNAIII showed very good correlation with the ratios of RNAIII versus gyrB RNA standard inputs and were therefore used as a simple readout to evaluate agr functionality. We validated the assay by characterizing 106 clinical S. aureus isolates, including strains with genetically characterized agr mutations. All isolates with dysfunctional agr activity exhibited a TTP ratio (TTP(gyrB)/TTP(RNAIII)) lower than 1.10, whereas agr-positive isolates had a TTP ratio higher than this value. The results showed that the assay was capable of determining target RNA ratios over 8 logs (10(-3) to 10(4)) with high sensitivity and specificity, suggesting the duplex NASBA assay may be useful for rapid determination of agr phenotypes and virulence potential in S. aureus clinical isolates.