Analysis of Environmental Mycobacterium Ulcerans

环境溃疡分枝杆菌分析

• 批准号: 7219178
• 负责人: RICHARD Ashley HURT
• 金额: $ 9.88万
• 依托单位: ATOM SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7219178
• 关键词: Area; Biological Assay; Complement; Country; DNA; DNA amplification; Detection; Development; Genome; Genus Mycobacterium; Human; Ligase; Ligation; Methods; Mycobacterium ulcerans; Numbers; Pathway interactions; Phase; Polymerase; Polymerase Chain Reaction; Process; Reaction; Relative (related person); Research; Research Personnel; Sampling; Sensitivity and Specificity; Site; Soil; Specificity; Techniques; Testing; Time; Ulcer; Urination; base; pathogen; skin disorder; transmission process

DESCRIPTION (provided by applicant): The aim of this research is to develop a locus-specific DNA amplification process suitable for highly multiplexed detection of pathogenic mycobacteria in environmental samples. Specifically, this project aims to develop an assay to identify the presence of Mycobacterium ulcerans (MU) in environmental samples to determine the transmission pathway from the environmental to humans. MU is the causative agent responsible for Buruli ulcer, a devastating skin disease present in several countries. The proposed approach is to create a polymerase chain reaction (PCR) template that contains PCR primer sites that are not present in the target genome but contains a specific sequence in the target genome. This is done by creating two site-specific probes, each one containing one of the PCR primer sites or a complement thereof. These probes anneal to the target DNA at each end of a sequence in the genome that contains only three of the four possible DNA bases. Polymerase is used to extend one of the probes across this void region so that the complement of the void region is created. Then ligase is used to connect this extension product to the other probe, creating the PCR template. This void-extension-ligation (VEL) reaction can be repeated many times making several copies of the template. PCR is then performed, amplifying the targeted region of the DNA. Because the PCR primers are introduced in the probes, a single set of PCR primers can be used for a large number of targeted sequences. Other researchers have shown that a similar process called MARA, which also can use a single set of PCR primers, can be multiplexed successfully for 750 separate targeted sequences across nine DNA samples. The proposed technique is somewhat simpler than MARA and should be even more specific. Specificity and multiplex capability are key features in targeting regions that are putatively unique to M. ulcerans but which, in fact, may exist in related mycobacteria in an environmental sample. In Phase I, we plan to develop the VEL-PCR method and test it on environmental samples inoculated with M. ulcerans and others inoculated with its closes relative, M. marinum. After sensitivity and specificity have been demonstrated in Phase I, actual samples from highly endemic areas will be analyzed. Further development of VEL-PCR will be performed in Phase II to adapt it to the detection of other important pathogens.

描述（由申请人提供）：本研究的目的是开发一种位点特异性 DNA 扩增方法，适用于环境样品中致病性分枝杆菌的高度多重检测。具体来说，该项目旨在开发一种检测方法来识别环境样本中溃疡分枝杆菌（MU）的存在，以确定从环境到人类的传播途径。 MU 是布鲁里溃疡的病原体，布鲁里溃疡是一种在多个国家存在的破坏性皮肤病。所提出的方法是创建一个聚合酶链式反应 (PCR) 模板，其中包含目标基因组中不存在的 PCR 引物位点，但包含目标基因组中的特定序列。这是通过创建两个位点特异性探针来完成的，每个探针包含一个 PCR 引物位点或其互补位点。这些探针在基因组序列的每一端与目标 DNA 退火，该序列仅包含四个可能的 DNA 碱基中的三个。 使用聚合酶将探针之一延伸穿过该空白区域，从而产生空白区域的互补部分。然后使用连接酶将该延伸产物连接到另一个探针，从而创建 PCR 模板。这种空隙延伸连接 (VEL) 反应可以重复多次，制作模板的多个副本。然后进行 PCR，扩增 DNA 的目标区域。由于PCR引物被引入探针中，因此一组PCR引物可用于大量目标序列。其他研究人员表明，称为 MARA 的类似过程也可以使用一组 PCR 引物，可以成功地对 9 个 DNA 样本中的 750 个单独的目标序列进行多重分析。所提出的技术比 MARA 稍微简单一些，并且应该更加具体。特异性和多重能力是靶向区域的关键特征，这些区域被认为是溃疡分枝杆菌独有的，但实际上可能存在于环境样品中的相关分枝杆菌中。 在第一阶段，我们计划开发 VEL-PCR 方法，并在接种了溃疡分枝杆菌的环境样本和接种了其近亲海分枝杆菌的其他样本上进行测试。在第一阶段证明敏感性和特异性后，将对来自高流行地区的实际样本进行分析。 VEL-PCR 的进一步开发将在第二阶段进行，以使其适应其他重要病原体的检测。