New Sensitive Detection of Food and Water Borne Pathogens

描述（由申请人提供）：疾病控制和预防中心（CDC）估计，在美国，每年有超过7500万疾病，30万住院治疗，每年有5000人死亡，直接归因于食源性病原体bbb。大肠杆菌O157:H7、单核增生李斯特菌、肠道沙门氏菌和细小隐孢子虫等食源性和水媒病原体每年造成100万例感染。美国疾病控制与预防中心估计，这些微生物的感染每年给美国造成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，我们的公共卫生系统面临的一个主要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方法，以灵敏地检测和鉴定与食物和水传播疾病有关的传染因子（细菌、病毒和原生动物寄生虫）。在第一阶段，我们打算开发一种基于耦合SNIPase探针和滚动环技术的荧光、多重、单管方法，以选择性地识别存在于感兴趣的特定致病微生物基因组中的定义目标序列。

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.