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Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

Gopaul KK, Sells J, Lee R, Beckstrom-Sternberg SM, Foster JT, Whatmore AM - BMC Res Notes (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK. Krishna.Gopaul@apha.gsi.gov.uk.

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.

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Related in: MedlinePlus

Melt curves of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the HRM melt curves directly. Curves move from right to left with decreasing DNA concentration. As shown, the curves generated by very low concentrations of B. abortus DNA are very close to the curves generated by high concentrations of B. melitensis DNA and could be misidentified through this association.
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Fig2: Melt curves of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the HRM melt curves directly. Curves move from right to left with decreasing DNA concentration. As shown, the curves generated by very low concentrations of B. abortus DNA are very close to the curves generated by high concentrations of B. melitensis DNA and could be misidentified through this association.

Mentions: To determine the sensitivity of discrimination of HRM assays, titrations of the genomic DNA from the five target Brucella species were prepared and tested. Through titrations it was determined that the limit for reproducible discrimination of the five individual HRM assays was 100 fg (data not shown). It was noted that whilst using the negative first derivative of the data, there was no difference in the positioning of melt peaks with changing DNA concentration (Figure 1), there were changes in the kinetics of the melt curve with DNA concentration (Figure 2). As interpretation of the quintuplex was performed visually (Figures 3 and 4), comparing the curve dynamic of the sample with that of a known control on the same run, low concentrations of non-target Brucella could be misinterpreted as high concentrations of the target species. In a review of HRM, Reed et al.[33] mentioned that interpretation of HRM data was better achieved through the comparison of melt curves rather than from the first derivative melt peak which was subject to “data smoothing”. It was for this reason that the decision was made to standardise the amount of DNA tested to 1 ng/μl in subsequent work.Figure 1


Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

Gopaul KK, Sells J, Lee R, Beckstrom-Sternberg SM, Foster JT, Whatmore AM - BMC Res Notes (2014)

Melt curves of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the HRM melt curves directly. Curves move from right to left with decreasing DNA concentration. As shown, the curves generated by very low concentrations of B. abortus DNA are very close to the curves generated by high concentrations of B. melitensis DNA and could be misidentified through this association.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307374&req=5

Fig2: Melt curves of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the HRM melt curves directly. Curves move from right to left with decreasing DNA concentration. As shown, the curves generated by very low concentrations of B. abortus DNA are very close to the curves generated by high concentrations of B. melitensis DNA and could be misidentified through this association.
Mentions: To determine the sensitivity of discrimination of HRM assays, titrations of the genomic DNA from the five target Brucella species were prepared and tested. Through titrations it was determined that the limit for reproducible discrimination of the five individual HRM assays was 100 fg (data not shown). It was noted that whilst using the negative first derivative of the data, there was no difference in the positioning of melt peaks with changing DNA concentration (Figure 1), there were changes in the kinetics of the melt curve with DNA concentration (Figure 2). As interpretation of the quintuplex was performed visually (Figures 3 and 4), comparing the curve dynamic of the sample with that of a known control on the same run, low concentrations of non-target Brucella could be misinterpreted as high concentrations of the target species. In a review of HRM, Reed et al.[33] mentioned that interpretation of HRM data was better achieved through the comparison of melt curves rather than from the first derivative melt peak which was subject to “data smoothing”. It was for this reason that the decision was made to standardise the amount of DNA tested to 1 ng/μl in subsequent work.Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK. Krishna.Gopaul@apha.gsi.gov.uk.

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.

Show MeSH
Related in: MedlinePlus