Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis

<p>Abstract</p> <p>Background</p> <p>Brucellosis, caused by members of the genus <it>Brucella</it>, remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family <it>Brucella </it>largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel <it>Brucella </it>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 <it>Brucella </it>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.</p> <p>Results</p> <p>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 <it>Brucella </it>spp and the marine mammal <it>Brucella</it>. Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and <it>Brucella </it>isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of <it>Brucella</it>, 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 <it>Brucella </it>from close phylogenetic relatives on the same platform.</p> <p>Conclusion</p> <p>The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of <it>Brucella </it>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 <it>Brucella </it>groups, to further type at the subspecies level, or to include markers for additional useful characteristics.</p

Differentiation of Tuberculosis Strains in a Population with Mainly Beijing-family Strains

A new panel of 25 VNTR-MIRU loci differentiates Beijing-family TB strains better than a panel of 15

Transcription of the Mycobacterium tuberculosis recA gene

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Characterization of the Two Mycobacterium tuberculosis recA Promoters

The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ(70) −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage

Rapid and Reliable Single Nucleotide Polymorphism-Based Differentiation of Brucella Live Vaccine Strains from Field Strains▿

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

Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo

1. We examined the relationship between oxidant stress and the vasodepressor activity of glyceryl trinitrate (GTN) in vivo, including rapid GTN tolerance development, in 13-week old obese and age-matched lean Zucker rats which had been maintained for 4 weeks on either control diet or diets enriched with the lipophilic, chain-breaking antioxidants vitamin E (0.5% w w(−1)) or probucol (0.5% w w(−1)) or the superoxide anion scavenger tiron (1% w v(−1) in drinking water). 2. The basal plasma level of the isoprostane 8-epi-PGF(2α), an in vivo marker of lipid peroxidation, was elevated by approximately 5 fold in the obese Zucker rat and markedly reduced by dietary lipophilic antioxidants and depressed by dietary tiron. 3. Vasodepression to bolus does GTN (0.1–100 μg kg(−1) i.v.), but not endothelium-dependent vasodepression to bolus dose acetylcholine (ACh, 0.02–2.0 μg kg(−1) i.v.), was impaired in obese animals and completely restored by dietary antioxidants. 4. Nitrate tolerance developed in vivo during a 1 h infusion of GTN (40 μg kg(−1) min(−1) i.v.) appeared more severe in obese animals. However, rapid nitrate tolerance was not affected by dietary antioxidants in either the obese or lean Zucker rat. 5. We therefore provide evidence that elevated oxidant stress in the obese Zucker rat is associated with an impairment in nitrate vasodepressor activity. However, our data are not consistent with either a role for oxidant stress in rapid nitrate tolerance development in the anaesthetized Zucker rat or the aggravation of this tolerance by pre-existing oxidant stress