Publications (7) View all
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Article: Mycobacterium tuberculosis H37Rv sigC is expressed from two promoters but is not auto-regulatory.
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ABSTRACT: The extracytoplasmic function (ECF) sigma factor SigC has been implicated in the pathogenesis of Mycobacterium tuberculosis but control of its expression and activity is poorly understood. No proteins that interact with SigC have been detected leading to the suggestion that this sigma factor may be primarily controlled at the level of transcription. It has been suggested that SigC may be autoregulatory and a role has also been proposed for SigF in the expression of sigC. In this study we identified two promoters that were active under standard growth conditions by a combination of transcript start site mapping and promoter-lacZ fusion assays. The dominant promoter, P1, closely resembled mycobacterial SigA-dependent promoters, and introduction of a single base change at the conserved A of the -10 region eliminated promoter activity. Although the sequence of the other, P2, closely resembled the reported SigC consensus motifs, expression directed by this promoter was unaltered in a ΔsigC mutant strain, or in strains defective in other ECF sigma factors for which some similarity in consensus sequences was apparent. Comparison of the effects of different changes in the -10 region suggested that the P2 promoter was most likely recognised by SigA.Tuberculosis (Edinburgh, Scotland) 01/2012; 92(1):48-55. · 2.54 Impact Factor -
Article: Rapid and reliable single nucleotide polymorphism-based differentiation of Brucella live vaccine strains from field strains.
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ABSTRACT: The reliable differentiation of live Brucella vaccine strains from field isolates is an important element in brucellosis control programs. We describe the design, validation, and implementation of a novel single nucleotide polymorphism (SNP)-based typing platform that offers a rapid, reliable, and robust tool to achieve this with improved diagnostic accuracy compared to existing molecular tests. Furthermore, the assays described are designed such that they supplement, and can be run as an intrinsic part of, a previously described assay identifying Brucella isolates to the species level (K. K. Gopaul, C. J. Smith, M. S. Koylass, and A. M. Whatmore, BMC Microbiol. 8:86), giving a comprehensive molecular typing platform.Journal of clinical microbiology 02/2010; 48(4):1461-4. · 4.16 Impact Factor -
Article: Comparative performance of SNP typing and 'Bruce-ladder' in the discrimination of Brucella suis and Brucella canis.
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ABSTRACT: Two novel molecular assays, 'Bruce-ladder' and SNP typing, have recently been described designed to differentiate isolates of the genus Brucella, causative organisms of the significant zoonotic disease brucellosis, at the species level. Differentiation of Brucella canis from Brucella suis by molecular approaches can be difficult and here we compare the performance of 'Bruce-ladder' and SNP typing in correctly identifying B. canis isolates. Both assays proved easy to perform but while 'Bruce-ladder' misidentifies a substantial proportion of B. canis isolates as B. suis, all B. canis isolates were correctly identified by SNP typing.Veterinary Microbiology 10/2009; 142(3-4):450-4. · 3.33 Impact Factor -
Article: Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis.
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ABSTRACT: Brucellosis, caused by members of the genus Brucella, remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family Brucella largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel Brucella have been reported from various marine mammals and voles. Classical identification to species level is based on a biotyping approach that is lengthy, requires extensive and hazardous culturing and can be difficult to interpret. Here we describe a simple and rapid approach to identification of Brucella isolates to the species level based on real-time PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus. Seven pairs of short sequence Minor Groove Binding (MGB) probes were designed corresponding to SNPs shown to possess an allele specific for each of the six classical Brucella spp and the marine mammal Brucella. Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and Brucella isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of Brucella, all of which typed as predicted when compared to other phenotypic and genotypic approaches. The assay is sensitive being capable of detecting and differentiating down to 15 genome equivalents. We further describe the design and testing of assays based on three additional SNPs located within the 16S rRNA gene that ensure positive discrimination of Brucella from close phylogenetic relatives on the same platform. The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of Brucella to the species level. Furthermore, being based on a robust phylogenetic framework, the assay provides a platform that can readily be extended in the future to incorporate newly identified Brucella groups, to further type at the subspecies level, or to include markers for additional useful characteristics.BMC Microbiology 01/2008; 8:86. · 3.04 Impact Factor -
Article: Progression toward an improved DNA amplification-based typing technique in the study of Mycobacterium tuberculosis epidemiology.
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ABSTRACT: While high-copy-number IS6110-based restriction fragment length polymorphism (HCN-RFLP) is the gold standard for typing most Mycobacterium tuberculosis strains, the time taken for culturing and low throughput make it impractical for large-scale prospective typing of large numbers of isolates. The development of a new method, mycobacterial interspersed repetitive units (MIRU), a variation of the original variable-number tandem repeat (VNTR) technique, may provide a viable alternative. Panels based on the original 12-loci MIRU (12MIRU), a combination of 12MIRU and remaining ETR loci (15MIRU-VNTR), and an extended panel with an additional 10 novel regions (25VNTR) were used to study three populations with varying degrees of epidemiological data. MIRU discrimination increased with panel size and the addition of spoligotyping. Combining these two techniques enabled a reduction in the panel size from 25 to 14 loci without a significant loss in discrimination. However, 25VNTR alone or in combination with spoligotyping still possessed weaker discrimination than RFLP for high-copy-number isolates.Journal of Clinical Microbiology 08/2006; 44(7):2492-8. · 4.15 Impact Factor
