Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species
| Autor: | Krishna K. Gopaul, Stephen M Beckstrom Sternberg, Jeffrey T. Foster, Robin Lee, Adrian M. Whatmore, Jessica Sells |
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| Rok vydání: | 2014 |
| Předmět: |
High Resolution Melting (HRM)
Single-nucleotide polymorphism Brucella Biology Real-Time Polymerase Chain Reaction Polymorphism Single Nucleotide General Biochemistry Genetics and Molecular Biology High Resolution Melt law.invention Species Specificity law Genotype Multiplex Typing Species identification Polymerase chain reaction Medicine(all) Genetics Whole genome sequencing Biochemistry Genetics and Molecular Biology(all) General Medicine biology.organism_classification Real time PCR Genes Bacterial Research Article |
| Zdroj: | BMC Research Notes |
| ISSN: | 1756-0500 |
| DOI: | 10.1186/1756-0500-7-903 |
| Popis: | Background The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Results Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. Conclusion The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-903) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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