Analysis of Environmental Mycobacterium Ulcerans

描述（由申请人提供）：本研究的目的是开发一种基因座特异性DNA扩增方法，适用于环境样本中致病性分枝杆菌的高度多重检测。具体而言，该项目旨在开发一种检测方法，以确定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几个国家存在的毁灭性皮肤病。所提出的方法是创建聚合酶链式反应（PCR）模板，其含有靶基因组中不存在但含有靶基因组中的特定序列的PCR引物位点。这通过产生两个位点特异性探针来完成，每个探针含有PCR引物位点之一或其互补物。这些探针在基因组中仅包含四种可能的DNA碱基中的三种的序列的每个末端与靶DNA退火。 聚合酶用于使探针之一延伸穿过该空隙区域，使得产生空隙区域的互补物。然后使用连接酶将该延伸产物连接到另一个探针，从而产生PCR模板。该空隙延伸连接（VEL）反应可以重复多次，产生模板的几个拷贝。然后进行PCR，扩增DNA的靶区域。由于PCR引物被引入探针中，因此单组PCR引物可用于大量靶序列。其他研究人员已经证明，一种类似的称为MARA的过程，也可以使用一组PCR引物，可以成功地对9个DNA样本中的750个单独的靶序列进行多重检测。所提出的技术比MARA简单一些，而且应该更加具体。特异性和多重性是靶向M.溃疡，但事实上，可能存在于环境样品中的相关分枝杆菌中。 在第一阶段，我们计划发展VEL-PCR方法，并在接种M. ulcerans等接种近缘种M.海产品。在第一阶段证明了灵敏度和特异性之后，将对来自高度流行地区的实际样本进行分析。VEL-PCR的进一步发展将在第二阶段进行，以适应其他重要病原体的检测。

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.