New Sensitive Detection of Food and Water Borne Pathogens

食品和水源性病原体的新灵敏检测

• 批准号: 6990807
• 负责人: Jay George
• 金额: $ 10万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990807
• 关键词: bacterial DNA; bacterial genetics; biohazard detection; bioterrorism /chemical warfare; fluorescence; food contamination; genetic strain; molecular probes; nucleic acid amplification techniques; nucleic acid sequence; technology /technique development

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.

描述（由申请人提供）：疾病控制与预防中心（CDC）估计，在美国，每年有超过7500万疾病，300,000次住院和5,000例死亡，这直接归因于食源性病原体[1]。大肠杆菌O157：H7，单核细胞增生李斯特氏菌，沙门氏菌肠和孢子虫的食物和水传播病原体每年对100万个感染负责[1]。疾病预防控制中心估计，这些生物体的感染每年耗资10亿美元，所有与食物和水有关的疾病总计50亿美元。因此，我们公共卫生系统的主要挑战是预防食物和水传播病原体引起的感染。该项目的目的是开发一种与食物和水传播疾病相关的传染剂（细菌，病毒和原生动物寄生虫）的敏感检测和鉴定的方法。在第1阶段，我们打算根据耦合的SNIPASE探针和滚动圆技术开发一种荧光，多路复用，单管方法，以选择性地识别存在的特定致病性微生物基因组中存在的定义的靶序列。 在该项目的第一阶段，我们建议： 使用来自大肠杆菌O157：H7的目标DNA序列作为模型，将SNIPASE探针与滚动圆扩增技术相结合。 证明将滚动圆技术和SNIPASE探针结合起来以区分大肠杆菌O157：H7和其他密切相关的菌株的可行性。 显示在荧光多重测定格式中检测病原体序列的可行性。 SNIPASE探针和滚动圆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。通过耦合这两种反应，我们预计我们可以在相对较短的时间内获得超过十亿倍扩增。此外，由于该技术的固有性质，因为没有3'OH结束的底漆，因此我们可以由于引物二聚体的形成而消除低信号与比率，以及与与其他多重技术相关的错误序列和随后的DNA合成相关的背景。