Using protein-DNA chimeras for HAT diagnosis

描述(申请人提供)：布氏冈比亚锥虫和罗德赛锥虫是人类非洲锥虫病(HAT)的病原体。如果不治疗，死于帽子是不可避免的。在患者常见的农村地区，在疾病的关键早期阶段，未能通过显微镜观察血液涂片和/或脑脊液中的锥虫可能是治疗失败的最重要因素。我们提出了一种新的技术，我们认为这将是重要的检测脑脊液中的寄生虫在第二阶段的疾病(中枢神经系统参与)，特别是在现场环境。这一过程使用了被称为“蝌蚪”的蛋白质-DNA嵌合体。为了创造“蝌蚪”，需要在蛋白质的单个位置添加一个“DNA条形码”。通过聚合酶链式反应扩增“蝌蚪”DNA，建立了具有很大动态范围和特异性的分析方法。然而，作为PCR的替代，我们建议通过环介导的等温扩增(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目标DNA。我们建议将LAMP技术与“蝌蚪”相结合，创建基于LAMPole的检测方法，用于HAT的诊断，适合现场诊断应用。有两个具体目标。它们是：1)设计寡核苷酸并创建用于高度灵敏地检测锥虫标志性蛋白的“LAMPoles”，以及2)确定LAMPoles在诊断2期HAT中的分析/临床敏感性和特异性。成功完成这项申请的具体目标将为早期发现适用于非洲保健中心和实地的第2阶段HAT提供新的诊断测试。公共卫生相关性：非洲人类锥虫病或昏睡病如果不治疗，是100%致命的。在非洲，每年至少有4万人死亡，因为在更多的农村地区，没有良好的诊断测试。使用简单的水浴，我们相信我们将开发的一种名为“LAMPoles”的新技术将为野外条件下的感染提供快速、准确的证据。

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.