Multiplexed Detection of Food and Waterborne Pathogens

食品和水源病原体的多重检测

• 批准号: 7685345
• 负责人: FRANCIS BARANY
• 金额: $ 87.83万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7685345
• 关键词: Antibiotic Resistance; Bacillus anthracis; Bacteria; Biological Assay; Blood; Blood capillaries; Brucella abortus; Calicivirus; Campylobacter jejuni; Capillary Electrophoresis; Categories; Centers for Disease Control and Prevention (U.S.); Clinical; Computer software; Cryptosporidium parvum; Culicidae; Cyclospora cayatanensis; DNA; Data Analyses; Dengue; Dengue Virus; Detection; Devices; Diarrhea; Disease; Disease Outbreaks; Encephalitozoon cuniculi; Entamoeba dispar; Entamoeba histolytica; Enterocytozoon bieneusi; Escherichia coli; Extended Family; Family; Feces; Fluorescence Resonance Energy Transfer; Food; Francisella tularensis; Generations; Genes; Ghana; Giardia lamblia; Grant; Haiti; Hepatitis A; Hepatitis A Virus; Housing; Infection; International; Isospora belli; Laboratories; Ligase; Liquid substance; Listeria monocytogenes; Medical center; Methods; Microbiology; Microfluidic Microchips; Microsporidia; Molecular; Molecular Profiling; National Institute of Allergy and Infectious Disease; Nested PCR; Norovirus; Organism; Patients; Performance; Plasma; Preparation; Protocols documentation; Protozoa; Puerto Rico; RNA Viruses; Reaction; Recombinant DNA; Red Cross; Research Personnel; Robotics; Rotavirus; Salmonella; Sampling; Sapovirus; Screening procedure; Sensitivity and Specificity; Septata intestinalis; Serotyping; Shigella; Simulate; Site; Specimen; System; Techniques; Testing; Toxin; Variant; Vibrio; Viral; Virulence; Virus; Virus Diseases; Water; Water Supply; West Nile virus; Yersinia enterocolitica; Yersinia pestis; biothreat; capillary; foodborne; meetings; microbial; pathogen; pathogenic Escherichia coli; prevent; programs; waterborne

DESCRIPTION (provided by applicant): The ability to rapidly detect food and water-borne pathogens is of utmost importance in preventing outbreaks associated with contamination of our nation's food and water supply. Existing detection systems have a limited ability to simultaneously screen a single sample for multiple agents. To meet this need we propose to use the ligase detection reaction (LDR) combined with PCR, and Universal Array detection, which we have already validated in identifying and distinguishing blood-borne bacterial and viral pathogens, including Category A biothreat agents. We will transfer these assays onto the Cepheid GeneXpert system, as well as evaluate their performance in modular microfluidic devices. The above techniques will be used to meet the specific aims of this application: Aim 1. To identify multiple food and waterborne bacteria simultaneously from a single sample. We will develop multiplexed and nested PCR/LDR assays to identify Category B bacterial pathogens (Campylocbacter jejuni, Yersinia enterocolitica, Salmonella spp, Shigella spp, Diarrheagenic E. coli, Pathogenic Vibrio spp, and Listeria monocytogenes). Stool specimens from patients with diarrheal disease will be evaluated in the microbiology laboratory and tested using the above molecular techniques. Random samples will also be spiked with DNA of uncommon bacterial pathogens to simulate infection with these agents. Aim 2. To identify multiple food and waterborne protozoa and viruses simultaneously from a single sample. PCR/LDR and RT-PCR/LDR will be used to detect the Category B food and water-borne protozoa (Cryptosporidium parvum, Cyclospora cayatanensis, Entamoeba histolytica, Giardia lamblia and Microsporidia) and viruses (Caliciviruses, Hepatitis A virus and Rotavirus) respectively. Stool specimens from patients with suspected protozoan or viral infections will be evaluated in the microbiology laboratory and tested using these molecular techniques. Aim 3. To develop high throughput methods for screening of multiple food and waterborne pathogens using molecular signature profiles. Standardized protocols using common liquid handling robotic platforms will be established using the techniques developed in Aims 1 and 2. The test will be validated for detecting bacterial pathogens, protozoa, and viruses from 3,000 stool samples. The PCR/LDR assays will be migrated to the Cepheid GeneXpert system as well as other microfluidic devices initially using capillary electrophoresis or LDR-FRET, and subsequently Universal Array readout.

描述（由申请人提供）：快速检测食物和水传播病原体的能力对于防止与国家食品和供水污染相关的爆发至关重要。现有检测系统的能力有限，可以同时筛选多种代理的单个样本。为了满足这一需求，我们建议将连接酶检测反应（LDR）与PCR和通用阵列检测结合使用，我们已经在识别和区分血液传播细菌和病毒病原体（包括生物疗法类别）方面已经验证了。我们将将这些测定法转移到头孢菌素Expert系统上，并评估它们在模块化微流体设备中的性能。上述技术将用于满足此应用程序的具体目的： 目的1。从单个样品同时识别多种食物和水传播细菌。我们将开发多路复用和嵌套的PCR/LDR分析，以鉴定B类细菌病原体（Campylocbacter Jejuni，Yersinia enterocolitica，Salmonella SPP，Shigella spp，Shigella SPP，腹泻性E. coli，致病性颤动纤维纤维SPP和单细胞基因生成菌）。将在微生物实验室中评估腹泻病患者的粪便标本，并使用上述分子技术进行了测试。随机样品还将用不常见的细菌病原体的DNA峰值，以模拟这些药物的感染。 目标2。从单个样品中同时鉴定多种食物和水传播原生动物和病毒。 PCR/LDR和RT-PCR/LDR将用于检测B类食物和水传播原生动物（隐孢子虫Parvum，Cyclospora cayatanensis，Entamoeba Histolytica，giardia lamblia and Micrososporidia）和病毒（caliciviruses a verus a verus an a verus and rotavirus and calivirus and calivirus and califirus and calivirus and calivirus and calvirus and rotavirus and calavirus and colivirus and rotavirus）均相应。将在微生物实验室中评估来自怀疑原生动物或病毒感染患者的粪便标本，并使用这些分子技术进行了测试。 目标3。开发使用分子特征剖面筛查多种食物和水传播病原体的高吞吐量方法。将使用AIMS 1和2中开发的技术建立使用常见液体处理机器人平台的标准化协议。该测试将经过验证，用于检测3,000个粪便样品的细菌病原体，原生动物和病毒。 PCR/LDR分析将迁移到Cepheid GenExpert系统以及最初使用毛细管电泳或LDR-FRET的其他微流体设备，然后是通用阵列读数。