Situm M, Grahovac B, Marković S, Lipozencić J, Poje G, Dobrić I, Marinović B, Bolanca-Bumber S, Misić-Majerus L
Department of Dermatovenerology, Zagreb University Hospital Center, Salata 4, 10000 Zagreb, Croatia.
Croat Med J. 2000 Mar;41(1):47-53.
To isolate and genotype Borrelia burgdorferi genospecies in serum samples of Croatian patients with erythema migrans.
DNA isolates from sera of patients with erythema migrans were analyzed by nested polymerase chain reaction (PCR), amplifying a segment of flagellin gene with primers encompassing the conserved region of the gene. To screen PCR products for heterogeneity, we performed single-stranded conformation polymorphism (SSCP) analysis. The samples showing differences in SSCP patterns were sequenced, and the sequence compared in the GeneBank for sequence homology with known Borrelia burgdorferi genospecies. We also constructed phylogenetic tree of all known borrelial sequences.
The nested PCR method using specially designed flagellin gene primers, achieved the sensitivity of 10 genome copies (0.01 pg of purified Borrelia burgdorferi DNA from culture) by dilution analysis. The assay specificity was confirmed by amplification of a part of the flagellin gene from different bacterial species. The primer pairs successfully amplified only Borrelia burgdorferi flagellin gene. The genome of Borrelia burgdorferi sensu lato was detected in the sera of all 10 tested patients with erythema migrans. Sequence data and phylogenetic analysis confirmed that all amplified samples belonged to Borrelia afzelii genospecies.
Phylogenetic tree analysis placed the borrelial isolates together with Borrelia afzelii sequences into a single group. This finding was additionally supported by sequence homology analysis, which produced a homology score of 99%. In patients with erythema migrans who come from the northwest Croatia, an endemic area for Lyme borreliosis, Borrelia afzelii was the cause of skin manifestations of Lyme borreliosis.
从克罗地亚患有游走性红斑的患者血清样本中分离伯氏疏螺旋体基因种并进行基因分型。
采用巢式聚合酶链反应（PCR）分析游走性红斑患者血清中的DNA分离物，用包含鞭毛蛋白基因保守区域的引物扩增该基因的一段片段。为筛选PCR产物的异质性，我们进行了单链构象多态性（SSCP）分析。对SSCP图谱显示有差异的样本进行测序，并将序列在基因库中与已知的伯氏疏螺旋体基因种进行序列同源性比较。我们还构建了所有已知疏螺旋体序列的系统发育树。
通过稀释分析，使用专门设计的鞭毛蛋白基因引物的巢式PCR方法达到了10个基因组拷贝（0.01 pg来自培养物的纯化伯氏疏螺旋体DNA）的灵敏度。通过扩增不同细菌物种的鞭毛蛋白基因的一部分来确认检测的特异性。引物对仅成功扩增了伯氏疏螺旋体鞭毛蛋白基因。在所有10名接受检测的游走性红斑患者的血清中均检测到伯氏疏螺旋体狭义基因组。序列数据和系统发育分析证实，所有扩增样本均属于阿氏疏螺旋体基因种。
系统发育树分析将疏螺旋体分离株与阿氏疏螺旋体序列归为同一组。这一发现得到了序列同源性分析的进一步支持，其同源性得分达到了99%。在克罗地亚西北部莱姆病的流行地区，患有游走性红斑的患者中，阿氏疏螺旋体是莱姆病皮肤表现的病因。
