Using protein-DNA chimeras for HAT diagnosis

使用蛋白质-DNA 嵌合体进行 HAT 诊断

• 批准号: 7932036
• 负责人: Dennis John Grab
• 金额: $ 20.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932036
• 关键词: Africa; Africa South of the Sahara; African; African Trypanosomiasis; Agglutination; Antibodies; Antigens; Area; Bathing; Biological Assay; Blood; Blood - brain barrier anatomy; Central Nervous System Diseases; Cerebrospinal Fluid; Cessation of life; Chimera organism; Clinical Sensitivity; Coma; DNA; DNA-Directed DNA Polymerase; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Drowsiness; Early Diagnosis; Environment; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Epidemic; Failure; Healthcare; Human; Humidity; Hybrids; Infection; Invaded; Investigation; Letters; Malaria; Mediating; Mental disorders; Methods; Monitor; Names; Nature; Neuraxis; Neurologic; Oligonucleotides; Parasites; Pathogen detection; Patients; Procedures; Proteins; Reaction; Seizures; Sensitivity and Specificity; Serological; Site; Sleeplessness; Specificity; Speed; Stage II Sleep; Staging; Tadpoles; Techniques; Technology; Temperature; Testing; Treatment Failure; Treatment Protocols; Tropical Disease; Trypanosoma; Trypanosoma brucei gambiense; Trypanosoma brucei rhodesiense; Trypanosomiasis; Vector-transmitted infectious disease; Visual; Water; Work; base; design; disease diagnosis; dosage; magnesium pyrophosphate; neglect; new technology; novel diagnostics; pathogen; public health relevance; rural area; success; working group

DESCRIPTION (provided by applicant): Trypanosoma brucei gambiense and T. b. rhodesiense are pathogens responsible for human African trypanosomiasis (HAT). Death from HAT is inevitable if untreated. In the rural areas where patients are typically seen, failure to microscopically observe trypanosomes in blood smears and/or CSF in the critical early stages of the disease is probably the single most important factor in failed treatment. We propose a new technology that we believe will be important for the detection of parasites in the CSF during Stage 2 disease (CNS involvement), particularly in the field setting. The procedure uses protein-DNA chimeras termed 'Tadpoles'. To create 'Tadpoles', a 'DNA barcode' is added to a single site on a protein. Amplification of the 'Tadpoles' DNA by PCR creates assays with great dynamic range and specificity. However, as an alternative to PCR we propose to amplify the DNA-barcode associated with Tadpoles by loop-mediated isothermal amplification (LAMP). The LAMP reaction amplifies DNA with high specificity, efficiency and speed under isothermal conditions and allows for easy visual positive identification of the target DNA. We propose to combine the technology of LAMP with 'Tadpoles' to create LAMP-based 'LAMPole' assays for the diagnosis of HAT suitable for diagnostic applications in the field. There are two Specific Aims. These are 1) to design oligonucleotides and create "LAMPoles" for the highly sensitive detection of trypanosome signature proteins, and 2) to define analytical/clinical sensitivity and specificity for LAMPoles in the diagnosis of Stage 2 HAT. Success in the completion of the Specific Aims of this application will provide novel diagnostic tests for the early detection of Stage 2 HAT applicable in African health care centers and in the field. PUBLIC HEALTH RELEVANCE: Human African trypanosomiasis or sleeping sickness is 100% fatal if not treated. At least 40,000 people die every year in Africa because good tests for diagnosis in the more rural areas do not exist. Using a simple water bath, we believe that a new technology we will develop called 'LAMPoles' will provide rapid, accurate, evidence for infection in field conditions.

描述（由申请人提供）：Trypanosoma brucei gambiense 和 T. b.罗得西亚锥虫是导致人类非洲锥虫病 (HAT) 的病原体。如果不及时治疗，HAT 导致的死亡是不可避免的。在经常见到患者的农村地区，在疾病的关键早期阶段未能用显微镜观察血涂片和/或脑脊液中的锥虫可能是治疗失败的最重要因素。我们提出了一种新技术，我们认为该技术对于第二阶段疾病（累及中枢神经系统）期间脑脊液中寄生虫的检测非常重要，特别是在现场环境中。该程序使用被称为“蝌蚪”的蛋白质-DNA 嵌合体。为了创造“蝌蚪”，需要将“DNA条形码”添加到蛋白质的单个位点上。通过 PCR 扩增“蝌蚪”DNA，可创建具有较大动态范围和特异性的检测方法。然而，作为 PCR 的替代方案，我们建议通过环介导等温扩增 (LAMP) 来扩增与蝌蚪相关的 DNA 条形码。 LAMP 反应在等温条件下以高特异性、高效率和高速度扩增 DNA，并可以轻松地目视阳性识别目标 DNA。我们建议将 LAMP 技术与“Tadpoles”相结合，创建基于 LAMP 的“LAMPole”测定法，用于诊断适合现场诊断应用的 HAT。有两个具体目标。这些是 1) 设计寡核苷酸并创建“LAMPole”，用于高度灵敏地检测锥虫特征蛋白，2) 定义 LAMPole 在 2 期 HAT 诊断中的分析/临床敏感性和特异性。成功完成本申请的具体目标将为非洲卫生保健中心和现场适用的第 2 阶段 HAT 的早期检测提供新颖的诊断测试。公共卫生相关性：非洲人类锥虫病或昏睡病如果不治疗的话 100% 致命。非洲每年至少有 40,000 人死亡，因为农村地区不存在良好的诊断测试。我们相信，使用简单的水浴，我们将开发的一项名为“LAMPoles”的新技术将为现场条件下的感染提供快速、准确的证据。