Differentiation of Staphylococcus spp. by high-resolution melting analysis

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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.

L’analyse de fusion à haute résolution HRMA (« high-resolution melting analysis ») est une méthode à haut débit rapide (post-PCR) qui permet de chercher les variations d’un gène cible. Le but de cette étude consistait à tester le potentiel de la HRMA pour distinguer des espèces bactriennes particulières du genre Staphylococcus même en utilisant une PCR à large spectre au sein du gène de l’ARNr 16S, où les différences de séquences sont mimines. Des échantillons d’ADN génomique isolés de 12 souches de référence du staphylocoque (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri et Staphylococcus xylosus) ont été soumis à une amplification par PCR en temps réel cibant le gène ARNr 16S en présence du colorant fluorescent EvaGreen™, suivie d’une HRMA. Les profils de fusion ont été utilisés comme empreinte moléculaire pour différencier les espèces bactériennes. La HRMA de S. saprophyticus et S. xylosus a généré des profils semblables car leurs séquences respectives de la région d’ARN r16S analysée sont identiques. Les autres souches de référence ont été parfaitement différenciées soit directement, soit par des tracés à haute résolution obtenus par la formation d’hétéroduplexes entre les produits PCR co-amplifiés des souches de staphylocoques testées et une souche non reliée d’un point de vue phylogénique.

Document Type: Research Article

Publication date: December 1, 2010

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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