Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. Hence, detection of Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is essential in various infections. The aim of this study was to design a melting curve analysis (MCA) assay based on Multiplex Real-Time PCR for rapid detection of staphylococci and antibiotic resistance. The current study used standard strains of positive and negative coagulase staphylococci. As the first step, serial dilutions were prepared with ratios of 108 to 101 cfu/mL based on standard bacterial concentration with 0.5 McFarland and the results were illustrated in dedicated melting curves. All melting curves of gene amplification had an equal melting point. In all dilutions, the observed melting temperatures shown in the melting curves of gene amplification were equal to 83.79°C for ITS-gene, 74.2°C for phop gene, 76.49°C for sap-gene, 78.2°C for mvaA gene, 79.57°C for 16srRNA-gene, 74.83°C for mupA gene, and 76.6°C for mecA gene. The MCA based on real time-PCR for identifying staphylococcal species and antibiotic resistance is a highly effective method with high sensitivity and specificity.