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

  • ️Tue Jan 01 2008

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

Krishna K Gopaul et al. BMC Microbiol. 2008.

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1

Seven Species defining MGB assay profile. Example of the multiple outcome assay used to identify twelve Brucella isolates. Each species-defining assay is run in rows (A-G) with samples run in columns. The green PCR profiles represent reactions with the VIC labelled probe, representing the species-specific probe in each probe pair. The blue PCR profiles represent reactions with the FAM labelled probes, representing the reaction with the alternate state (non species-specific) allele probe in each probe pair. The identity of each of the isolates 1–12 is indicated by a red dot where an isolate generates a positive PCR reaction with a VIC-labelled probe.

Figure 2
Figure 2

Discrimination of the seven species defining assays. Allele discrimination plots generated by each species-defining MGB assay when applied to 303 Brucella isolates. Each assay was read after 40 cycles.

Figure 3
Figure 3

Sensitivity and limits of discrimination using MGB assay. Sensitivity of the species defining assays. The data presented here relates to the B. melitensis gap assay with the remaining six assays giving equivalent results. A. Titrations of B. melitensis 16 M DNA from 5 ng to 500 ag. B. Allele discrimination plot showing performance of the B. melitensis assay in distinguishing B. melitensis from non-B. melitensis (B. suis) down to 50 fg of DNA.

Figure 4
Figure 4

Alignments of various 16SrRNA sequences around the location of the 16S rRNA771/778 probes. Alignments of fragments of 16S rRNA centred around SNPs at bases 771 and 778 relative to B. abortus 9–941, used in combination with the 16S rRNA1055 SNP to separate Brucella from closely related bacteria. The alignment contains 65 sequences taken directly from Genbank and includes many examples of Ochrobactrum, the closest phylogentic neighbour of Brucella as well as other, less-closely related, members of the α-proteobacteria. In this figure, the targetted Brucella specific SNPs are highlighted in red and the non-Brucella alternatives in blue. The green bar above both figures represents the location of probe hybridisation. The alignment includes only one Brucella sequence as there is 100% identity in the 16SrRNA sequences between all Brucella [34]. Using this assay on its own will discriminate most but not all of the non- Brucella shown from Brucella organisms.

Figure 5
Figure 5

Alignments of various 16SrRNA sequences around the location of the 16S rRNA1055 probes. Alignments of fragments of 16S rRNA centred around SNP at base 1055 relative to B. abortus 9–941 showing location of the SNP, used in combination with the 16S rRNA771/778 SNPs to separate Brucella from closely related bacteria. The alignment contains 65 sequences taken directly from Genbank and includes many examples of Ochrobactrum, the closest phylogentic neighbour of Brucella as well as other, less-closely related, members of the α-proteobacteria. In this figure, the targetted Brucella specific SNP is highlighted in blue and the non-Brucella alternatives in red. The green bar above both figures represents the location of probe hybridisation. The alignment includes only one Brucella sequence as there is 100% identity in the 16SrRNA sequences between all Brucella [34]. Whilst this assay on its own is not as discriminatory as 16S rRNA771/778 assay, it crucially distinguishes non-Brucella not detected by the afore mentioned assay.

Figure 6
Figure 6

Discrimination of the Brucella genus defining assays. Application of the two 16S rRNA based probe pairs in distinguishing isolates from the genus Brucellae from other α-proteobacteria. a. Results generated by the 16SrRNA771/778 probe pair when used against 5 species of Ochrobactrum (including three strains of O. anthropi) and representatives of all the known Brucella species and biovars. This reaction separates Brucella isolates from all other except O. anthropi ATCC49237. b. Results generated with the application of 16SrRNA1055 probe pair illustrating the use of this assay to further separate O. anthropi ATCC49237 from Brucella isolates.

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References

    1. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, Garin-Bastuji B, Letesson JJ. From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet Res. 2005;36:313–326. doi: 10.1051/vetres:2005003. - DOI - PubMed
    1. Moreno E, Cloeckaert A, Moriyón I. Brucella evolution and taxonomy. Vet Microbiol. 2002;90:209–227. doi: 10.1016/S0378-1135(02)00210-9. - DOI - PubMed
    1. Michaux-Charachon S, Bourg G, Jumas-Bilak E, Guigue-Talet P, Allardet-Servent A, O'Callaghan D, Ramuz M. Genome structure and phylogeny in the genus Brucella. J Bacteriol. 1997;179:3244–3249. - PMC - PubMed
    1. Whatmore AM, Perrett LL, MacMillan AP. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol. 2007;7:34. doi: 10.1186/1471-2180-7-34. - DOI - PMC - PubMed
    1. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, Cutler SJ, MacMillan AP. Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol. 2006;44:1982–1993. doi: 10.1128/JCM.02039-05. - DOI - PMC - PubMed

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