aureus genotyping can be easily combined with the interrogation of other genetic markers. This single- and closed-tube single-step method for S. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.Īnalysis of the spa locus by HRM resolves spa sequence variants. Real-time quantitative RT-PCR was performed on a Rotor Gene 6000 instrument (Corbett, Life Sciences, Australia) for the P53 gene (TaqMan Gene Expression Assays no. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. The 2 unresolved HRM spa types differed by only 1 bp. HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs. HRM curve comparison between runs was done to determine the portability of the method. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X. Relative expressions of IFNG and IFNG-AS1 genes were assessed in tumoral tissues and paired ANCTs in the rotor gene 6000 Corbett Real-Time PCR System. We describe here a method for genotyping S. Setting of a new temperature profile and running a cycler 1.1. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. Temperature profile setup To set the temperature profile, follow the manufacturer’s instructions for use (IFU).
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