Using protein-DNA chimeras for HAT diagnosis

描述（由申请人提供）：布氏冈比亚锥虫和罗得西亚锥虫是导致非洲人类锥虫病（HAT）的病原体。如果不及时治疗，死于HAT是不可避免的。在通常看到患者的农村地区，在疾病的关键早期阶段，未能在血液涂片和/或脑脊液中显微镜观察到锥虫，可能是治疗失败的一个最重要因素。我们提出了一种新的技术，我们相信这将是重要的检测寄生虫在脑脊液阶段2疾病（CNS累及），特别是在现场设置。这个过程使用了被称为“蝌蚪”的蛋白质- dna嵌合体。为了创造“蝌蚪”，在蛋白质上的一个位点上添加了一个“DNA条形码”。通过PCR扩增“蝌蚪”DNA创建具有很大动态范围和特异性的分析。然而，作为PCR的替代方法，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，并且可以很容易地对目标DNA进行视觉阳性鉴定。我们建议将LAMP技术与“蝌蚪”技术相结合，创建基于LAMP的“LAMPole”检测方法，用于诊断HAT，适用于该领域的诊断应用。有两个具体目标。这些是1)设计寡核苷酸并创建用于高灵敏度检测锥虫特征蛋白的“lampole”，以及2)确定lampole诊断2期HAT的分析/临床敏感性和特异性。成功完成这一应用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二阶段艾滋病提供新的诊断测试。公共卫生相关性：如果不治疗，非洲人类锥虫病或昏睡病是100%致命的。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检测。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.