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Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs).

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Bottom Line: The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk.The method is rapid and the results are reproducible.HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

Background: Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.

Results: An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.

Conclusion: This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

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Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.
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Figure 3: Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.

Mentions: Amplified products were sized by electrophoresis on 3% Metaphor or 4% agarose gels such that the 8-bp increase of each additional repeat unit was identified by comparison with a 25-bp mass ladder. Sample data of the amplicons generated from three loci of the B. abortus biovar type strains and two laboratory strains, are shown in Fig. 3A. Alternatively, the HOOF-Print procedure was adapted for high throughput, automated fluorescent DNA fragment analysis, by tagging each unique upstream primer with one of three fluorescent labels (Table 2). The amplicon sizes were determined by comparison with a co-migrating DNA mass ladder labeled with an alternative fluorescent tag. Data generated for VNTR Locus-1 by automated fluorescent DNA fragment analysis for the same isolates assayed in Fig. 3A, are shown in Fig. 3B. Detection of the fluorescent tagged amplicons was typically very sensitive with a high signal to noise ratio. Accurate sizing of the amplicons was essential for calculating the number of repeat units at each locus.


Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs).

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183870&req=5

Figure 3: Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.
Mentions: Amplified products were sized by electrophoresis on 3% Metaphor or 4% agarose gels such that the 8-bp increase of each additional repeat unit was identified by comparison with a 25-bp mass ladder. Sample data of the amplicons generated from three loci of the B. abortus biovar type strains and two laboratory strains, are shown in Fig. 3A. Alternatively, the HOOF-Print procedure was adapted for high throughput, automated fluorescent DNA fragment analysis, by tagging each unique upstream primer with one of three fluorescent labels (Table 2). The amplicon sizes were determined by comparison with a co-migrating DNA mass ladder labeled with an alternative fluorescent tag. Data generated for VNTR Locus-1 by automated fluorescent DNA fragment analysis for the same isolates assayed in Fig. 3A, are shown in Fig. 3B. Detection of the fluorescent tagged amplicons was typically very sensitive with a high signal to noise ratio. Accurate sizing of the amplicons was essential for calculating the number of repeat units at each locus.

Bottom Line: The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk.The method is rapid and the results are reproducible.HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

Background: Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.

Results: An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.

Conclusion: This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

Show MeSH
Related in: MedlinePlus