LYME DISEASE DIAGNOSIS BY PCR/DNA PROBE SYSTEM

目前用于诊断莱姆病的方法取决于 血清学检测和都不够敏感的检测 早期疾病和在后期阶段不够具体。 直接 检测病原体伯氏疏螺旋体使用敏感的 而特异性测定似乎是这些问题的唯一解决方案。 一 DNA探针检测结合扩增程序，如聚合酶 链反应（PCR）可以提供极端的特异性和灵敏度， 以及提供关于生物体本身的有价值的信息，例如。 菌株之间的变异（流行病学数据）。 在欧洲，晚期 莱姆病最常与神经系统后遗症或 慢性皮肤病，而在N.美国，最常见的联想是 莱姆关节炎 这种差异可能是由于重要的差异 菌株之间。 B。burgdorferi已经证明自己是一个高度 通过血清学和DNA同源性的异质性生物群 问题研究 到目前为止，设计能够检测所有的PCR系统的尝试是失败的。 分离的B。由于这一点，burgdorferi取得了有限的成功， 基因组变异性 使用高度保守的基因，如核糖体 RNA基因，未能提供必要的特异性。 但是现有 P.I.的研究已经表明另一个高度保守的基因ie.的 鞭毛蛋白基因是一个潜在的候选者， 侦测 这些初步研究表明，该系统能够 都能检测到B。burgdorferi特异性和鉴别B型B。 burgdorferi菌株分成至少三个不同的纲。 拟议研究的主要目的将是开发这一系统 直接检测莱姆病的特异性和灵敏度测定 同时能够区分B的疾病。布格多尔费里-into 流行病学显著群体。 将进行实验， 证明PCR/DNA探针系统能够扩增 从B的所有致病性分离株中提取合适的DNA片段。burgdorferi 而不扩增来自近亲的相应DNA片段 人类致病性疏螺旋体属的物种。 此外，实验将 进行测试的探针序列，已开发的 B分化。burgdorferi分为不同的类。 的序列 将针对来自北美的大量菌株进行测试， 以确定日本和欧洲的最大分组数 存在于B中。burgdorferi。 这项工作将与 开发探针序列。 任何新的和不寻常的菌株将 通过克隆和测序详细研究了相关 其鞭毛蛋白基因的片段。 将努力建立一个 各组与疾病表现的相关性，即。神经系统 对比关节炎后遗症。 还将进行实验，以全面评估敏感性 引物/探针系统的最终配置。 此外该 引物/探针系统检测微量 螺旋体已经被添加到不同量的外来的 将评估污染的核酸。 这项工作的目的是 确定试验灵敏度的理论水平， 模拟真实的患者样本的条件。 问题 还将为各种患者标本提供最佳样品制备， 处理。 将被考虑的样品将包括：皮肤穿孔 活检、心肌活检、血液、脑脊液、滑液 和尿液。 最后，将进行实验以证明 引物/探针系统快速诊断莱姆病的临床应用 使用PCR和真实的患者样本来检测疾病。

Current methods used for the diagnosis of Lyme disease depend upon serologic testing and are both insufficiently sensitive for the detection of early disease and insufficiently specific in the later stages. Direct detection of the causative organism Borrelia burgdorferi using a sensitive and specific assay appears to be the only solution to these problems. A DNA probe test coupled to amplification procedures such as the polymerase chain reaction (PCR) could provide extreme specificity and sensitivity as well as providing valuable information about the organism itself, eg. variation between strains (epidemiological data). In Europe, late-stage Lyme disease has most often been associated with neurological sequelae or chronic skin disease whereas in N. America, the most usual association is with Lyme arthritis. This disparity could be due to important differences between strains. B. burgdorferi has shown itself to be a highly heterogeneous group of organisms by both serologic and DNA homology studies. Thus far, attempts to design PCR systems capable of detecting all isolates of B. burgdorferi have met with limited success due to this genomic variability. The use of highly conserved genes, such as ribosomal RNA genes, has failed to provide the necessary specificity. However, prior studies by the P.I. have shown that another highly conserved gene ie. the flagellin gene is a potential candidate for both specific and sensitive detection. These preliminary studies have shown that the system is capable of both detecting B. burgdorferi specifically and differentiating B. burgdorferi strains into at least three distinct classes. The principal aim of the proposed research will be to develop this system into a specific and sensitive assay for the direct detection of Lyme disease which is at the same time able to differentiate B. burgdorferi into epidemiologically significant groups. Experiments will be undertaken to demonstrate that the PCR/DNA probe system is capable of amplifying an appropriate segment of DNA from all pathogenic isolates of B. burgdorferi without amplifying a corresponding segment of DNA from closely related species of human pathogenic Borrelia. In addition, experiments will be undertaken to test the probe sequences that have been developed for the differentiation of B. burgdorferi into distinct classes. The sequences will be tested against a very large number of strains from North America, Japan and Europe in order to determine the maximum number of sub-groups present within B. burgdorferi. This will proceed simultaneously with the development of probe sequences. Any new and unusual strains will be investigated in a detailed by cloning and sequencing of the relevant section(s) of their flagellin gene. Attempts will be made to establish a correlation between the groups and disease manifestation ie. neurologic versus arthritic sequelae. Experiments will also be undertaken to exhaustively assess the sensitivity of the primer/probe system in its final configuration. In addition, the ability of the primer/probe system to detect minute quantities of spirochetes which have been added to varying quantities of extraneous contaminating nucleic acids will be assessed. The aim of this work will be to determine the theoretical level of sensitivity of the assay under conditions which mimic those of real patient specimens. The question of optimal sample preparation for a variety of patient specimens will also be addressed. Samples which will be considered will include:skin punch biopsies, myocardial biopsies, blood, cerebro-spinal fluid, synovial fluid and urine. Finally, experiments will be carried out to demonstrate the clinical utility of the primer/probe system for the rapid diagnosis of Lyme disease using PCR and real patient samples.