Ultra-Sensitive Molecular Diagnostic for Borrelia Species

描述（由申请人提供）：拟议研究的目的是开发一种使能技术，该技术将允许开发一种分子诊断方法，用于鉴定和区分密切相关的细菌物种，这些细菌物种在低微生物数量下导致重大疾病。这项技术将通过开发一种诊断系统来检测伯氏疏螺旋体的种类，特别强调孤独伯氏疏螺旋体，这种细菌在美国东南部9个州从人类身上移除的约1.2%的孤星蜱中被发现。孤星蜱与一种叫做“南方蜱相关皮疹病”（STARI）的疾病有关，这种疾病与莱姆病类似。然而，可能是因为缺乏诊断测试，许多患有STARI的患者中没有发现B. lonestari。在建议的诊断中，使用限制性内切酶消化来自患者血液（或其他标本）的DNA以产生携带一个或多个物种特异性特征序列的小DNA片段。然后加热来分离这两条DNA链。接下来是一个反应，使用一种酶特异性地连接单链DNA片段的末端，单链DNA片段的每一端都有一个特定的序列，形成一个包含病原体特定序列的单链DNA环。然后进行一种称为滚环扩增的反应，以制造一条长DNA链，这是环形DNA补体的串联重复。第二段DNA用于启动超支链位移扩增反应，在不到90分钟的时间内产生109倍的特定核酸片段。在第二阶段，我们将把这种诊断扩展到更多的伯氏疏螺旋体物种，并让一个经过认证的实验室执行第一阶段对人类患者样本开发的测试程序，以确定STARI的来源。这项工作的一个附带好处将是对莱姆病的敏感诊断。公共卫生相关性：拟议的工作将开发一种可行的分子技术，用于诊断来自引起人类疾病的微生物的极低拷贝数DNA。有些细菌，如疏螺旋体属的种类，在低拷贝数时就能引起重大疾病。拟议的研究将产生一个诊断系统，可以同时检测莱姆病和所有其他疑似引起人类疾病的伯氏疏螺旋体。拟议的研究还试图发现“南方蜱虫相关皮疹病”（STARI）的原因。莱姆病是北美最常见的病媒传播疾病，自1982年以来报告的病例超过20万例。2002年，发病率为每10万人8.2例，约95%的病例发生在东北部、中北部和大西洋中部各州。在美国南部，另一种疏螺旋体（Borrelia lonestari）已被证明可引起莱姆病样症状（STARI）。目前尚不清楚STARI的发病率，因为没有分子或血清学诊断测试来确定B. lonestari和相关伯氏疏螺旋体的感染。如果非莱姆病病原体的伯氏疏螺旋体是STARI的病因，那么美国东南部持续感染的患者目前仍未得到诊断和治疗。

DESCRIPTION (provided by applicant): The aim of the proposed research is to develop an enabling technology that will allow development of a molecular diagnostic for identification and discrimination among closely related bacterial species that cause significant disease at low microorganism numbers. The technology will be advanced through the development of a diagnostic system for detection of species of Borrelia with special emphasis on the species Borrelia lonestari, a bacterium which has been found in about 1.2% of the Lone Star ticks removed from humans in nine southeastern US states. Lone Star ticks are associated with a condition called "southern tick associated rash illness" (STARI), which is similar to Lyme disease. However, B. lonestari has not been found in many patients suffering from STARI, perhaps because of the lack of diagnostic tests. In the proposed diagnostic, DNA from a patient's blood (or other specimen) is digested using a restriction enzyme to generate small DNA fragments carrying one or more species specific signature sequences. Heat is then used to separate the two strands of DNA. This is followed by a reaction that uses an enzyme to specifically join the ends of single stranded DNA fragments that have a specific sequence at each end to form a single "stranded DNA circle containing the pathogen" specific sequence. A reaction called rolling circle amplification is then performed to make a long DNA strand that is a tandem repeat of the circular DNA's complement. A second section of DNA is used to initiate a hyper branched strand displacement amplification reaction, yielding a 109 fold increase in the specific nucleic acid fragment in less than 90 minutes. In Phase II, we will extend this diagnostic to additional Borrelia species and have a certified laboratory perform the test procedure that was developed in Phase I on samples from human patients to determine the source of STARI. A fringe benefit of this work will be a sensitive diagnostic for Lyme disease. PUBLIC HEALTH RELEVANCE: The proposed work would develop an enabling molecular technology for diagnosis of extremely low copy-number DNA from microorganisms that cause disease in humans. Some bacteria such as species of the genus Borrelia are able to cause significant disease at low copy" number. The proposed research will produce a diagnostic system that can simultaneously test for Lyme disease and all other Borrelia species suspected of causing disease in humans. The proposed research also seeks to discover the cause of "southern tick associated rash illness" (STARI). Lyme borreliosis is the most common vector borne disease in North America with more than 200,000 cases reported since 1982. In 2002, the incidence rate was 8.2 cases per 100,000 with approximately 95% of the cases occurring in the northeast, north central and mid Atlantic states. In the southern United States an additional species, Borrelia lonestari, has been shown to cause Lyme disease like symptoms (STARI). The incidence rate of STARI is currently unknown, because there is no molecular or serological diagnostic test to identify infection with B. lonestari and related Borrelia species. If Borrelia species other that the causative agent of Lyme disease are the cause of STARI, patients in the southeastern US that have a persistent infection currently remain undiagnosed, and untreated.