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，用于在微生物数量较少的情况下对引起重大疾病的密切相关细菌进行鉴定和区分。这项技术将通过开发一种检测疏螺旋体物种的诊断系统来推进，其中特别强调疏螺旋体物种，这种细菌在美国东南部9个州从人类身上取出的约1.2%的孤星蜱中发现。孤星蜱与一种名为“南方硬虱相关皮疹疾病”(STARI)的疾病有关，这种疾病类似于莱姆病。然而，在许多患有STARI的患者中并没有发现孤独巴氏杆菌，这可能是因为缺乏诊断测试。在拟议的诊断中，使用限制酶消化患者血液(或其他样本)的DNA，以产生携带一个或多个物种特异性签名序列的小DNA片段。然后用热量来分离这两条DNA。紧随其后的是使用一种酶专门连接单链DNA片段的末端的反应，这些单链DNA片段的每一端都有一个特定的序列，以形成一个单一的“包含病原体的链DNA环”的特定序列。然后进行一种名为滚环扩增的反应，以产生一条长的DNA链，这是环状DNA互补的串联重复。第二段DNA被用来启动超支链置换扩增反应，在不到90分钟的时间里，特定核酸片段增加了109倍。在第二阶段，我们将把这项诊断扩展到更多的疏螺旋体物种，并让一家经认证的实验室对第一阶段开发的人类患者样本进行测试程序，以确定STARI的来源。这项工作的一个附带好处是对莱姆病的敏感诊断。与公共卫生相关：这项拟议的工作将开发一种使能诊断极低拷贝数DNA的分子技术，这些DNA来自导致人类疾病的微生物。一些细菌，如疏螺旋体属的物种，能够在低拷贝数下引起重大疾病。这项拟议的研究将产生一种诊断系统，可以同时检测莱姆病和所有其他被怀疑会导致人类疾病的疏螺旋体物种。这项拟议的研究还试图发现“南方硬蜱相关皮疹疾病”(STARI)的原因。莱姆疏螺旋体病是北美最常见的媒介传播疾病，自1982年以来报告了20多万例。2002年，发病率为每100,000例中有8.2例，大约95%的病例发生在大西洋东北部、北部、中部和中部各州。在美国南部，另一个物种，疏螺旋体，已被证明会引起莱姆病样症状(STARI)。STARI的发病率目前尚不清楚，因为目前还没有分子或血清学诊断试验来确定感染孤独芽孢杆菌和相关疏螺旋体物种。如果莱姆病的病原体以外的疏螺旋体是STARI的原因，那么美国东南部持续感染的患者目前仍然没有得到诊断和治疗。

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.