High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus

Stephens, Alexander; Inman-Bamber, John; Giffard, Philip M.; Huygens, Flavia

doi:10.1373/clinchem.2007.093658

Cited by 70 publications

(62 citation statements)

References 18 publications

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“…Such hypervariable genes are potentially amenable to HRM analysis. This has been demonstrated with the Campylobacter jejuni CRISPR and fla loci (12) and the S. aureus spa locus (19,22). Those studies demonstrated that HRM can resolve significant numbers of alleles of individual loci.…”

Section: Figmentioning

confidence: 87%

Microbiological Applications of High-Resolution Melting Analysis

2012

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High-resolution melting (HRM) analysis uses real-time PCR instrumentation to interrogate DNA sequence variation and is a low-cost, single-step, closed-tube method. Here we describe HRM technology and provide examples of varied clinical microbiological applications to highlight the strengths and limitations of HRM analysis. The term "high-resolution melting" (HRM) analysis encompasses DNA fragment analysis techniques that involve the accurate determination of the relationship between temperature and extent of denaturation. First described by Carl Wittwer's group for clinical applications (6,8), HRM is typically performed with a real-time PCR instrument, immediately after the PCR. HRM is often used to interrogate specific single nucleotide polymorphisms (SNPs) but can also be applied to SNP discovery, the discrimination of alleles defined by multiple SNPs or tandemrepeat number, and the determination of DNA methylation status. The advantages of HRM technology are its low cost, use of generic instrumentation available in many laboratories, and the simplicity of the approach-with the great majority of applications effectively incorporating single-step and "closed-tube" methods.While whole-genome sequencing is likely to supersede many microbial genotyping methods, the speed and simplicity of HRM and its generic technology platform suggest that microbiological analysis protocols that incorporate HRM for first-pass screening may have considerable utility. Indeed, as high-throughput sequencing improves the knowledge of specific mutations correlated to important microbiological phenotypes, there will be more targets for HRM-based assays. The growing popularity of HRM for microbiological applications is reflected by the 125 publications in this field since 2006, with 59 publications in the past 18 months alone (to 8 June 2012). Here we briefly describe HRM technology and provide examples of clinical microbiological applications to highlight the strengths and limitations of HRM. HRM THEORY AND TECHNOLOGYThe denaturation of a DNA fragment with increasing temperature defines the "melting curve." This is usually sigmoidal in shape (Fig. 1), and the melting temperature (T m ) can be calculated by determining the peak of the melting curve first derivative (dF/dT). The T m is positively correlated with sequence length and percent GC content (%GϩC). An approximation of the relationship between sequence content and length is as follows: T m ϭ 81.5 ϩ 16.69 ϫ (log[Na ϩ ]) ϩ 0.41 ϫ (%GϩC) Ϫ (500/sequence length). The correlation of T m with %GϩC is due to the additional hydrogen bond between GC pairs compared with AT pairs. According to the formula given above, the T m change (⌬T m ) corresponding to a single G-C ↔ A-T change is (41/sequence length) in°C. We have extensive experience with the Corbett Rotorgene device, and in our hands the limit for robust ⌬T m discrimination is approximately 0.2°C. This is the ⌬T m predicted from a single G-C ↔ A-T change in a 205-bp sequence. Unsurprisingly, our experience is that 200 bp is the appr...

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Section: Figmentioning

confidence: 87%

Microbiological Applications of High-Resolution Melting Analysis

2012

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“…It is also no more complex or labour intensive to perform than a simple PCR; it does not require purification and/or cloning of the amplicons, nor enzyme digestion or gel electrophoresis for single-strand conformation polymorphism, nor any kind of hybridization with probes on a solid support. In optimal conditions, it has been demonstrated that HRM analysis has almost 100% accuracy (Reed and Wittwer, 2004) and it has previously been adapted for several microbiological applications such as rapid identification of Staphylococcus aureus genotypes and influenza subtypes (Lin et al, 2008;Stephens et al, 2008). In addition, HRM has been applied in scanning human genome DNA for several disease causing polymorphisms (reviewed in Erali et al, 2008), while at the Rudjer Boskovic Institute it has previously been Sabol, I., Čretnik, M., Hadžisejdić, I., Si-Mohamed, A., Matovina, M., Grahovac, B., Levanat, S., Grce, M., 2009.…”

Section: Discussionmentioning

confidence: 99%

A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis

Sabol¹,

Čretnik²,

Hadžisejdić³

et al. 2009

Journal of Virological Methods

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A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis. Journal of Virological Methods 162, 142-147. doi:10.1016Methods 162, 142-147. doi:10. /j.jviromet.2009 This study showed that HRM analysis can be useful for the identification of HPV 16 variants. The HRM method will be useful in low resource settings as it saves considerable time and resources compared to sequencing.Key words: human papillomavirus 16; variant; high resolution melting; sequencing Abbreviations: HPV -human papillomavirus; PCR -polymerase chain reaction; HRM -high resolution melting; SNP -single nucleotide polymorphisms Sabol, I., Čretnik, M., Hadžisejdić, I., Si-Mohamed, A., Matovina, M., Grahovac, B., Levanat, S., Grce, M., 2009. A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis.

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“…HRM analysis of 44 diverse MRSA isolates carried out by Stephens and coworkers generated 20 profiles from 22 spa sequence types. The two unresolved HRM spa types differed by only 1 bp (Stephens et al, 2008). Surprisingly good results of genotyping of C. jejuni were obtained by HRM scanning of polymorphism of several targets: a regularly interspaced short-palindromic-repeat (CRISPR) locus (Price et al, 2007) 75 2010).…”

Section: High Resolution Melting Analysis -Hrmmentioning

confidence: 99%