LYME DISEASE DIAGNOSIS BY PCR/DNA PROBE SYSTEM

目前用于莱姆病诊断的方法依赖于 血清学检测，两者对检测都不够敏感 早期疾病，后期不够具体。直接 用一种灵敏的方法检测伯氏疏螺旋体 而特定的检测方法似乎是解决这些问题的唯一办法。一个 DNA探针检测与聚合酶等扩增程序相结合 链式反应(PCR)可提供极高的特异性和敏感性 并提供关于有机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，处于后期阶段 莱姆病最常与神经后遗症或 慢性皮肤病，而在北美，最常见的联系是 患有莱姆关节炎。这种差异可能是由于重要的差异造成的 在菌株之间。B.burgdorferi已经证明了自己是一种高度 通过血清学和DNA同源性确定的异质性生物群 学习。到目前为止，试图设计能够检测所有 因此，伯氏杆菌的分离株仅取得了有限的成功。 基因组的可变性。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。然而，之前 P.I.的研究表明，另一个高度保守的基因，即。这个 鞭毛蛋白基因是一种既特异又敏感的潜在候选基因 侦测。这些初步研究表明，该系统有能力 无论是对伯氏杆菌的特异性检测还是对伯氏杆菌的鉴别。 伯氏杆菌至少可分为三个不同的类别。 拟议研究的主要目标将是开发该系统 建立了一种特异而灵敏的直接检测莱姆病的方法 一种同时能够将伯氏杆菌区分为 具有重要流行病学意义的群体。将进行实验，以 证明该PCR/DNA探针系统能够扩增出 所有伯氏杆菌致病菌株的适当DNA片段 而不会从密切相关的DNA中扩增出相应的DNA片段 人类致病性疏螺旋体的种类。此外，实验将是 负责测试已经为 伯氏假单胞菌分化为不同的纲。这些序列 将对来自北美的大量菌株进行测试， 日本和欧洲，以确定分组的最大数量 存在于B.burgdorferi体内。这将与 开发探针序列。任何新的和不寻常的菌株都将是 通过克隆和测序对相关基因进行了详细的调查 鞭毛蛋白基因片段(S)。将尝试建立一个 各组别与疾病表现的相关性。神经学 对关节炎后遗症的治疗。 还将进行实验，以详尽地评估灵敏度 底漆/探头系统的最终配置。此外， 底物/探针系统检测微量的能力 螺旋体被添加到不同数量的外来 将对污染的核酸进行评估。这项工作的目的将是 确定该检测方法的理论灵敏度水平。 模拟真实病人标本的条件。这个问题是 对各种患者样本的最佳样品制备也将是 地址。将考虑的样品包括：蒙皮冲压机 活组织检查，心肌活组织检查，血液，脑脊液，滑液 还有尿液。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 疾病使用聚合酶链式反应和真实的患者样本。

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.