Bacterial Pathogen Amplification & Real-Time Detection

描述（申请人提供）：众所周知，生物恐怖主义在美国是一个现实。然而，除了难以安全生长和传播的炭疽、鼠疫耶尔森氏菌和天花等A类病原体外，还存在可用于感染我们的食物或供水的B类病原体。这些生物体包括细菌病原体、原生动物和病毒。除了这些天然的致病生物外，它们还可以通过基因工程来增加其毒力或抵抗标准的抗生素治疗。因此，非常需要用于快速敏感食品和水传播病原体检测的新方法，特别是如果它们也可以用于鉴定这些生物体内的药物敏感性。利用食品病原体的生物恐怖主义不仅仅是对美国的假设威胁。1984年秋天，俄勒冈州达拉斯的一个宗教邪教组织污染了杂货店和餐馆的食物，导致至少751人患病。该小组只是培养了他们从当地科学供应室获得的食物病原体鼠伤寒沙门氏菌，并将培养物撒在杂货店和餐馆沙拉吧的农产品上。如果该组织使用了一种更致命的病原体，如伤寒沙门氏菌，许多人肯定会死亡。该计划的总体目标是开发一种集成的等温DNA扩增和探针阵列检测载玻片，能够快速识别各种食品和水传播的病原体。所有NIAID生物防御类别B食品和水传播的细菌病原体E。大肠杆菌、霍乱弧菌、痢疾志贺氏菌、沙门氏菌、单核细胞增生李斯特菌、空肠弯曲杆菌和小肠结肠炎耶尔森菌将在该项目中检测。提出了一种能够在封闭的密封装置中实时等温扩增和检测所有这些生物体的单个集成载玻片。该程序还可以区分活的生物体和因食物或水卫生过程而死亡的生物体。此外，该测试可用于确定病原体的抗生素敏感性，以确定基因改变的生物体。

DESCRIPTION (provided by applicant): As we all know, bio-terrorism in America is a reality. However in addition to the Category A agents like anthrax, Yersinia pestis and smallpox, which are difficult to safely grow and disseminate, exist the Category B agents that could be used to infect our food or water supply. These organisms include bacterial pathogens, protozoa, and viruses. In addition to these natural pathogenic organisms they could also be genetically engineered to increase their virulence or to resist standard antibiotic treatments. Therefore new methods for rapid sensitive food and waterborne pathogen detection are greatly needed, especially if they can also be used to identify drug sensitivity within these organisms. Bio-terrorism using a food pathogen is not just a hypothetical threat to America. A religious cult in Dalles Oregon sickened at least 751 people by contaminating food in grocery stores and restaurants in the fall of 1984. The group simply grew cultures of the food pathogen Salmonella typhimurium that they obtained from their local scientific supply house and sprinkled the cultures on produce in the grocery stores and the restaurant salad bars. If the group had used a more deadly pathogen like Salmonella typhi that causes typhoid fever many people would certainly have died. The overall goal of this program is to develop an integrated isothermal DNA amplification and a probe array detection slide capable of rapidly identifying a variety of food and waterborne pathogens. All of the NIAID Biodefense Category B food and waterborne bacterial pathogens E. coli, Vibrio cholera, Shigella dysentery, Salmonella species, Listeria monocytogenes, Camphylobacter jejuni, and Yersinia enterocolitica will be detected in this program. A single integrated slide capable of isothermal amplifying and detecting all of these organisms in real-time in a closed sealed device is proposed. The program can also distinguish live organisms from dead organisms killed by the food or water sanitation process. Also, the test can be used to identify the antibiotic sensitivity of the pathogen to identify genetically altered organisms.