PMC3884711_01
PMC3884711_02
Detection and identification of Brucella DNA. A) Detection of Brucella DNA by quantitative PCR. Numbers indicate amplification curves with cycle threshold (Ct) values representative of samples. Curve 1, sample from patient 1 with 16 Ct value; curve 2, sample from patient 2 with 16 Ct; curve 3, sample from patient 3 with 17 Ct; curve 4, stem cells of cord blood from patient 1 with 34 Ct; curve 5, stem cells of cord blood from patient 2 with 34.5 Ct; curve 6, positive control with 18 Ct, curve 7, negative control with no Ct. B) Amplification of Brucella DNA by AMOS-PCR. Numbers indicate the amplified DNA bands representative of samples. Lane 1, DNA molecular weight marker, values along the left side are base pairs; lane 2, sample from patient 1; lane 3, sample from patient 2; lane 4, sample from patient 3; lane 5, stem cells from cord blood of patient 1; lane 6, stem cells from cord blood of patient 2; lane 7, negative control.
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F1
A). Additionally, the specific DNA bands for B. melitensis were identified from each patient's blood culture by an abbreviated B. abortus, melitensis, ovis, and suis (AMOS) PCR but were not observed from the twins' cord blood, possibly due to low levels of Brucella DNA (Figure 1, panel B).
Genetic relationship between the strain isolated in this study (BRU-TZ) and other Brucella melitensis strains. The variable number of tandem repeats were obtained for phylogenetic analysis at multiple-locus variable-number tandem repeat analysis bank version-4 (http://mlva.u-psud.fr) (,). The phylogenetic tree was plotted on the differences in variable number of tandem repeats at 16 loci obtained by multiple-locus variable-number tandem repeat analysis.
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F2
They were all genotyped as 16 loci, with variable number of tandem repeats of 1 5 3 13 2 3 3 2 6 22 9 6 9 11 4 5, which was phylogenetically closer to #20081716 and #9900139 strains prevalent in Spain (Figure 2) but differed from strains prevalent in Kyrgyzstan.