Portnoï Denis, Sertour Natacha, Ferquel Elisabeth, Garnier Martine, Baranton Guy, Postic Danièle
Laboratoire des Spirochètes, Institut Pasteur, Paris, France.
FEMS Microbiol Lett. 2006 Jun;259(1):35-40. doi: 10.1111/j.1574-6968.2006.00249.x.
Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.
莱姆病螺旋体病是由伯氏疏螺旋体狭义种（B. burgdorferi sl）复合体中的螺旋体引起的最重要的媒介传播疾病。有充分证据表明，这一组基因多样的螺旋体的不同物种与该疾病的不同临床表现有关。为了区分该细菌复合体中的物种，我们开发了一种针对hbb基因的实时PCR方案。我们设计了一种荧光素标记的探针，该探针特异性针对该基因中与物种相关的多态性区域。一个内部用Red640标记的引物使得荧光共振能量转移得以发生。该方法的灵敏度为每次检测10个细菌。扩增后，生成熔解曲线用于基因分型。对这些熔解曲线的分析清楚地实现了对欧洲主要的B. burgdorferi sl物种的区分。通过这种基于hbb的方法对170份蜱虫提取物进行了分析，并同时通过扩增5S - 23S基因间隔区和限制性片段长度多态性分析进行分析。这两种方法之间存在良好的相关性。我们得出结论，这种基于hbb的实时PCR适用于对野外采集的蜱虫进行流行病学研究，尽管探针所覆盖的基因组序列中的罕见突变可能导致错误鉴定。
