BACKGROUND: The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X. METHODS: The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. 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 curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs. RESULTS: HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method. CONCLUSION: Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.
BACKGROUND: The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X. METHODS: The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. 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 curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs. RESULTS: HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method. CONCLUSION: Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.
Authors: Shu-Biao Wu; Michelle G Wirthensohn; Peter Hunt; John P Gibson; Margaret Sedgley Journal: Theor Appl Genet Date: 2008-09-10 Impact factor: 5.699
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Authors: Shreema Merchant-Patel; Patrick J Blackall; Jillian Templeton; Erin P Price; Steven Y C Tong; Flavia Huygens; Philip M Giffard Journal: Appl Environ Microbiol Date: 2009-11-20 Impact factor: 4.792