Ultra-Sensitive Molecular Diagnostic for Borrelia Species

伯氏疏螺旋体物种的超灵敏分子诊断

• 批准号: 7481939
• 负责人: RICHARD Ashley HURT
• 金额: $ 7.33万
• 依托单位: ATOM SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481939
• 关键词: Amblyomma; American Type Culture Collection; Bacteria; Bite; Black-legged Tick; Blood; Blood Cells; Borrelia; Borrelia burgdorferi; Borrelia lonestari; Case Study; Cell Count; Cells; Circular DNA; Clinical; Complement; Complement component C5; Complex; Condition; Cytolysis; DNA; DNA Restriction Enzymes; DNA amplification; DNA copy number; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Digestion; Discrimination; Disease; Dissociation; Enzymes; Exanthema; Fringe Benefit; Genes; Genomics; Geographic state; Heating; Human; Incidence; Infection; Laboratories; Left; Letters; Ligase; Ligation; Link; Lyme Disease; Magnetic Bead Technology; Maps; Methods; Mitochondria; Molecular; Natural Killer Cells; North America; Nucleic Acids; Numbers; Oligodeoxyribonucleotides; Pathology; Patients; Phase; Procedures; Process; Public Health; Rate; Reaction; Research; Research Ethics Committees; Rickettsia; Sampling; Serological; Single-Stranded DNA; Source; Specimen; Spheroplasts; Staging; Symptoms; System; T4 DNA Ligase; Tandem Repeat Sequences; Technology; Testing; Ticks; United States; Vector-transmitted infectious disease; Work; Yeasts; base; design; member; microbial; microorganism; pathogen; relapsing fever borrelia; restriction enzyme

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.

描述（由申请人提供）：拟议研究的目的是开发一种使能技术，该技术将允许开发分子诊断技术，用于识别和区分在低微生物数量下引起严重疾病的密切相关的细菌种类。该技术将通过开发检测伯氏疏螺旋体（Borrelia）物种的诊断系统来推进，特别重点关注孤内疏螺旋体（Borrelia lonestari）物种，在美国东南部九个州从人体中取出的孤星蜱中约 1.2% 中发现了这种细菌。孤星蜱与一种称为“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 STARI 患者中并未发现 B.lonestari。在所提出的诊断中，使用限制性内切酶消化来自患者血液（或其他样本）的 DNA，以生成携带一个或多个物种特异性特征序列的小 DNA 片段。然后利用热量来分离两条 DNA 链。随后进行反应，使用酶特异性连接单链 DNA 片段的末端，这些片段的两端都有特定的序列，形成单个“含有病原体的 DNA 链环”特定序列。然后进行称为滚环扩增的反应，生成一条长 DNA 链，该链是环状 DNA 互补体的串联重复序列。第二段 DNA 用于启动超支链置换扩增反应，在不到 90 分钟的时间内将特定核酸片段扩增 109 倍。在第二阶段，我们将把这种诊断扩展到其他疏螺旋体属物种，并让经过认证的实验室对人类患者的样本执行第一阶段开发的测试程序，以确定 STARI 的来源。这项工作的一个附带好处是可以灵敏地诊断莱姆病。公共健康相关性：拟议的工作将开发一种可行的分子技术，用于诊断引起人类疾病的微生物中的极低拷贝数 DNA。一些细菌，例如伯氏疏螺旋体属的物种，能够以低拷贝数引起严重疾病。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疑似引起人类疾病的疏螺旋体属物种。拟议的研究还试图发现“南方蜱相关皮疹病”(STARI)的原因。莱姆疏螺旋体病是北美最常见的媒介传播疾病，有超过 200,000 例病例 自1982年以来就有报道。2002年，发病率为每10万人中有8.2例，其中约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，另一种物种孤独疏螺旋体已被证明会引起莱姆病样症状 (STARI)。目前 STARI 的发病率尚不清楚，因为没有分子或血清学诊断测试来识别 B. lonestari 和相关疾病的感染 伯氏疏螺旋体属。如果莱姆病病原体以外的疏螺旋体属物种是 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。