Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1-12 |
| Number of pages | 12 |
| Journal | BMC Research Notes |
| DOIs | |
| State | Accepted/In press - Dec 11 2014 |
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Keywords
- Brucella
- High Resolution Melting (HRM)
- Real time PCR
- Species identification
- Typing
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Medicine(all)
Cite this
Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species. / Gopaul, Krishna K.; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M.
In: BMC Research Notes, 11.12.2014, p. 1-12.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species
AU - Gopaul, Krishna K.
AU - Sells, Jessica
AU - Lee, Robin
AU - Beckstrom-Sternberg, Stephen M
AU - Foster, Jeffrey T
AU - Whatmore, Adrian M.
PY - 2014/12/11
Y1 - 2014/12/11
N2 - 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.
AB - 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.
KW - Brucella
KW - High Resolution Melting (HRM)
KW - Real time PCR
KW - Species identification
KW - Typing
UR - http://www.scopus.com/inward/record.url?scp=84928814788&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84928814788&partnerID=8YFLogxK
U2 - 10.1186/1756-0500-7-903
DO - 10.1186/1756-0500-7-903
M3 - Article
SP - 1
EP - 12
JO - BMC Research Notes
JF - BMC Research Notes
SN - 1756-0500
ER -