title: Differentiation of Staphylococcus spp. by high-resolution melting analysis.
abstract: 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.
authors.affiliation: Centre for Cardiovascular Surgery and Transplantations, Pekarska 53, 656 91 Brno, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Slany
authors.forename: Martina
authors.initials: M
authors.lastname: Vanerkova
authors.forename: Eva
authors.initials: E
authors.lastname: Nemcova
authors.forename: Barbora
authors.initials: B
authors.lastname: Zaloudikova
authors.forename: Filip
authors.initials: F
authors.lastname: Ruzicka
authors.forename: Tomas
authors.initials: T
authors.lastname: Freiberger
mesh_terms: D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020180:Heteroduplex Analysis; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013210:Staphylococcus / Q000145:classification* / Q000235:genetics*; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S