Reverse Line Blot Assay for Trypanosome and Leishmania Detection and Identificati

描述(申请人提供)：由锥虫家族成员引起的寄生虫病威胁着全世界5亿人，估计任何时候都会造成3000万人感染，在世界受影响的地区造成巨大的公共卫生和经济损失。这些疾病包括非洲锥虫病(昏睡病)、美洲锥虫病(恰加斯病)和利什曼病。非洲锥虫病在20世纪上半叶取得重大成功后死灰复燃；由于移民模式，恰加斯病已经蔓延到拉丁美洲最初的地理边界之外，现在威胁着美国；利什曼病已经从一种外来疾病转变为一种越来越频繁地被从中东返回美国的人员带回家的疾病。为了满足更好和更准确的寄生虫检测和鉴定的技术和操作需要，我们建议开发一种分子鉴定方法，可以在适当的实验室环境中由受过相应水平培训的人员进行。即将建立的锥虫反向杂交(Tryp-RLB)分析将包括两个顺序步骤：使用来自寄生虫基因组保守区的引物进行聚合酶链式反应扩增，然后针对排列在膜上的种、亚种或菌株特异性探针进行杂交。膜将由共价连接在大阵列中的寡核苷酸制备，并包装在一次性多通道培养盒中，允许每次分析运行多个PCR扩增样本的并行杂交。Tryp-RLB分析将在流通式抽吸装置中进行，该装置既加快了分析周转时间，又通过将扩增产物收集在密封烧瓶中防止了污染。获得了当天的检测结果，这比基于培养的方法和许多其他分子方法都有相当大的优势。本文将建立的鉴定锥虫和利什曼原虫的Tryp-RLB方法可应用于流行病学和诊断学以及治疗学、疫苗和其他基础研究领域的各种问题。它的格式允许它在选择膜大阵列的靶序列时在程序和应用上都是通用的，提供了一个在锥虫生物学领域前所未有的强大分析工具。 公共卫生相关性：非洲人类锥虫病、美洲锥虫病(恰加斯病)和利什曼病直接影响美洲、亚洲和非洲流行地区的3000多万人，而感染风险是这个数字的十倍。这些疾病的诊断和流行病学监测一直是具有挑战性的，因为引起这些疾病的寄生虫的生命周期复杂，很难识别感染症状，以及许多病原体物种之间的交叉反应。该项目将导致开发一种分子检测方法，用于检测和鉴定引起锥虫病和利什曼病两种形式的寄生虫物种和菌株。比目前方法更早和更准确的检测和识别将使临床医生能够为患者选择更有效的抗菌治疗，并将有助于流行病学监测和监测这些寄生虫的传播。

DESCRIPTION (provided by applicant): Parasitic diseases caused by members of the trypanosome family threaten five hundred million people worldwide and are responsible for an estimated 30 million infections at any time, representing an enormous public health and economic toll in affected regions of the world. These diseases include African trypanosomiasis (sleeping sickness), American trypanosomiasis (Chagas' disease) and leishmaniasis. African trypanosomiasis is resurgent after having been contained with significant success in the first part of the 20th century; Chagas' disease has spread beyond the original geographic boundaries of Latin America due to patterns of immigration and now threatens the U.S., and leishmaniasis has moved from an exotic disease to one which has been brought home at increasing frequency by personnel returning to the U.S. from the Mideast. To address the technical as well as operational needs for better and more accurate parasite detection and identification, we propose to develop a molecular identification assay that can be performed in a modest laboratory environment by personnel with corresponding level of training. The trypanosome reverse line blot (Tryp-RLB) assays to be developed will comprise two sequential procedures: Polymerase chain reaction amplification using primers derived from conserved regions within the parasite genomes, followed by hybridization against species, subspecies or strain-specific probes arrayed on a membrane. The membrane will be prepared with oligonucleotides covalently attached in a macroarray and packaged in a disposable multichannel incubation cassette, permitting parallel hybridization of multiple PCR-amplified samples per assay run. The Tryp-RLB assay will be carried out in a flow-through aspiration device which both accelerates the assay turnaround time and prevents contamination by collecting amplification products in a sealed flask. Same day assay results are obtained, a considerable advantage over both culture-based and many other molecular methods. The Tryp-RLB assay to be developed herein for identification of trypanosome and Leishmania species and strains can be applied to a wide variety of problems in areas of epidemiology and diagnostics as well as therapeutics, vaccines and other basic research. Its format allows it to be both generic in procedure and application specific in choice of target sequences for the membrane macroarray, providing a powerful analytical tool which is without precedent in the field of trypanosome biology. PUBLIC HEALTH RELEVANCE: Human African trypanosomiasis, American trypanosomiasis (Chagas' disease) and leishmaniasis directly affect over 30 million people in endemic regions of the Americas, Asia and Africa, while ten times that number are at risk of infection. Diagnosis and epidemiological monitoring of these diseases has been challenging due to the complex life cycles of the parasitic agents causing them, the difficulty in identifying symptoms of infection, and the cross-reactivities between many pathogen species. This project will result in the development of a molecular assay for detection and identification of the parasitic species and strains responsible for both forms of trypanosomiasis and leishmaniasis. Earlier and more accurate detection and identification than is possible with current methods will enable clinicians to select more effective antimicrobial therapy for patients, and will contribute to epidemiological monitoring and surveillance of the spread of these parasitic agents.