Development and Evaluation of Duplex MIRA-qPCR Assay for Simultaneous Detection of Staphylococcus aureus and non-aureus Staphylococci

Staphylococcus spp., especially Staphylococcus aureus (S. aureus), is an important pathogen in hospital-acquired infection and food poisoning. Here, we developed a multienzyme isothermal rapid amplification combined with duplex quantitative PCR (duplex MIRA-qPCR) method, which can simultaneously detect the S. aureus species-specific conserved gene FMN-bgsfp and the Staphylococcus genus-specific conserved gene tuf. This assay enabled the amplification of DNA within 20 min at a constant temperature of 39 °C. Specificity analysis indicated that all nine common Staphylococcus species were positive and non-Staphylococcus spp. were negative for tuf gene, whereas S. aureus was positive, non-aureus Staphylococci species and non-Staphylococcus spp. were negative for FMN-bgsfp gene, suggesting that duplex MIRA-qPCR exhibited high specificity. Meanwhile, the sensitivity was tested and the limit of detection (LoD) was 3 × 10(2) CFU/mL. The coefficient variation values ranged from 0.13% to 2.09%, indicating that the assay had good repeatability. Furthermore, all the nine common Staphylococcus species (including S. aureus) could be detected from four kinds of simulated samples and the LoD of S. aureus was 8.56 × 10(3) CFU/mL. In conclusion, the duplex MIRA-qPCR has advantages of stronger specificity, lower detection threshold, shorter detection time, and simpler operation, which is an effective tool to detect S. aureus and non-aureus Staphylococci spp. infections rapidly.