Differentiation of Staphylococcus spp. by high-resolution melting analysis
Language English Country Canada Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
21164574
DOI
10.1139/w10-091
PII: w10-091
Knihovny.cz E-resources
- MeSH
- DNA, Bacterial chemistry MeSH
- DNA Fingerprinting methods MeSH
- Species Specificity MeSH
- Heteroduplex Analysis MeSH
- Molecular Sequence Data MeSH
- DNA, Ribosomal chemistry genetics MeSH
- RNA, Ribosomal, 16S chemistry genetics MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Sequence Alignment MeSH
- Sensitivity and Specificity MeSH
- Staphylococcus classification genetics MeSH
- Transition Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Bacterial MeSH
- DNA, Ribosomal MeSH
- RNA, Ribosomal, 16S MeSH
High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.
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