New Sensitive Detection of Food and Water Borne Pathogens

描述(申请人提供)：疾病控制和预防中心(CDC)估计，在美国，每年有超过7500万例疾病、30万例住院和5000例死亡直接归因于食源性病原体[1]。O157：H7大肠杆菌，单核细胞增生性李斯特氏菌，肠沙门氏菌和微小隐孢子虫，食物和水传播的病原体每年导致100万人感染[1]。美国疾病控制与预防中心估计，这些微生物的感染每年给国家造成10亿美元的损失，所有与食物和水有关的疾病总共造成50亿美元的损失。因此，我们公共卫生系统面临的一个重大挑战是预防由食物和水传播的病原体引起的感染。该项目的目标是开发一种灵敏的检测和鉴定与食物和水传播疾病相关的传染病病原体(细菌、病毒和原生动物寄生虫)的方法。在第一阶段，我们打算开发一种基于耦合SNIPase探针和滚环技术的荧光、多重、单管方法，以选择性地识别存在于特定病原微生物基因组中的特定靶序列。 在该项目的第一阶段，我们建议： 以大肠杆菌O157：H7的靶DNA序列为模型，将SNIPase探针与滚环扩增技术相结合。 论证了滚环技术和SNIPase探针相结合用于区分大肠杆菌O157：H7和其他密切相关菌株的可行性。 展示以荧光多重分析形式检测病原体序列的可行性。 SNIPase探针和滚环扩增技术本身都没有必要的灵敏度来检测低水平的DNA靶标。通过将这两个反应结合起来，我们预计可以在相对较短的时间内实现超过10亿倍的放大。此外，由于这项技术的固有性质，即不存在大量具有3‘OH端的引物，因此我们可以消除由于引物二聚体形成而导致的低信噪比，以及与错误引发和随后的DNA合成相关的背景，与其他多重技术相关。

DESCRIPTION (provided by applicant): The Centers for Disease Control and Prevention (CDC) estimate that, in the United States, there are in excess of 75 million illnesses per year, 300,000 hospitalizations, and 5,000 deaths annually that are directly attributed to food-borne pathogens [1]. E. coli O157:H7, Listeria monocytogenes, Salmonella enterica and Cryptosporidium parvum food and water borne pathogens are accountable for 1 million infections annually [1]. The CDC estimates that infections from these organisms costs the country 1 billion dollars per year and 5 billion dollars total for all food and water related illness. Thus a major challenge to our public health system is the prevention of infections resulting from food- and water-borne pathogens. The goal of this project is to develop a method for the sensitive detection and identification of infectious agents (bacteria, viruses, and protozoan parasites) associated with food and water-borne diseases. In Phase 1, we intend to develop a fluorescent, multiplex, one-tube method based upon coupling SNIPase probes and rolling circle technology to selectively recognize defined target sequences that are present in the genome of specific pathogenic microorganisms of interest. In the first phase of this project we propose to: Combine SNIPase probes to Rolling Circle Amplification technology using target DNA sequences from E. coli O157:H7 as a model. Demonstrate the feasibility of combining rolling circle technology and SNIPase probes in to discriminate between E. coli O157:H7 and other closely related strains. Show the feasibility of detecting pathogen sequences in fluorescent multiplex assay formats. Neither SNIPase probes nor rolling circle amplification technology by themselves have the necessary sensitivity to detect low levels of DNA targets. By coupling the two reactions we anticipate that we can achieve greater than a billion fold amplification in a relatively short period of time. Additionally due to the inherent nature of the technology in that there is not an abundance of primers that have 3'OH ends, therefore we can eliminate low signal to ratios as a result of primer dimer formation as well background associated with the mis-priming and subsequent DNA synthesis associated with other multiplex technologies.