Using protein-DNA chimeras for HAT diagnosis

性状（由申请方提供）：冈比亚布氏锥虫和T. B.罗得西亚锥虫是引起人类非洲锥虫病（HAT）的病原体。如果不治疗，HAT的死亡是不可避免的。在患者常见的农村地区，在疾病的关键早期阶段未能在显微镜下观察到血涂片和/或CSF中的锥虫可能是治疗失败的最重要因素。我们提出了一种新的技术，我们认为这将是重要的寄生虫在CSF中的检测阶段2疾病（中枢神经系统的参与），特别是在现场设置。该程序使用称为“蝌蚪”的蛋白质-DNA嵌合体。为了创建“蝌蚪”，将“DNA条形码”添加到蛋白质上的单个位点。通过PCR扩增“蝌蚪”DNA产生具有大动态范围和特异性的测定。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，并允许容易地目视阳性鉴定靶DNA。我们建议将LAMP技术与“蝌蚪”技术联合收割机，以创建适用于该领域诊断应用的用于HAT诊断的基于LAMP的“LAMPole”测定。有两个具体目标。这些是1）设计寡核苷酸并创建用于锥虫特征蛋白的高灵敏度检测的“LAMPole”，和2）定义LAMPole在诊断2期HAT中的分析/临床灵敏度和特异性。成功完成本申请的特定目标将为早期检测适用于非洲卫生保健中心和现场的2期HAT提供新的诊断测试。公共卫生相关性：人类非洲锥虫病或昏睡病如果不治疗是100%致命的。在非洲，每年至少有4万人死亡，因为在农村地区没有良好的诊断测试。使用一个简单的水浴，我们相信，我们将开发一种名为“LAMPoles”的新技术，将在现场条件下提供快速，准确的感染证据。

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.