Multiplexed Detection of Food and Waterborne Pathogens

食品和水源病原体的多重检测

• 批准号: 7325231
• 负责人: FRANCIS BARANY
• 金额: $ 88.91万
• 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/7325231
• 关键词: Bacteria; Biological Assay; Blood; Calicivirus; Campylobacter jejuni; Capillary Electrophoresis; Categories; Centers for Disease Control and Prevention (U.S.); Clinical; Cryptosporidium parvum; Culicidae; Cyclospora cayatanensis; DNA; Dengue; Dengue Virus; Detection; Devices; Diarrhea; Disease; Disease Outbreaks; Encephalitozoon cuniculi; Entamoeba dispar; Entamoeba histolytica; Enterocytozoon bieneusi; Escherichia coli; Feces; Fluorescence Resonance Energy Transfer; Food; Generations; Genes; Ghana; Giardia lamblia; Haiti; Hepatitis A; Hepatitis A Virus; Infection; International; Isospora belli; Laboratories; Ligase; Liquid substance; Listeria monocytogenes; Medical center; Methods; Microbiology; Microfluidic Microchips; Microsporidia; Molecular; Molecular Profiling; Nested PCR; Norovirus; Organism; Patients; Performance; Plasma; Polymerase Chain Reaction; Protocols documentation; Protozoa; Puerto Rico; RNA Viruses; Reaction; Recombinant DNA; Red Cross; Ribosomal DNA; Robotics; Rotavirus; Salmonella; Sampling; Sapovirus; Screening procedure; Sensitivity and Specificity; Septata intestinalis; Serotyping; Shigella; Simulate; Site; Specimen; System; Techniques; Testing; Variant; Vibrio; Viral; Virulence; Virus; Virus Diseases; Water; Water Supply; West Nile virus; Yersinia enterocolitica; biothreat; foodborne; pathogen; pathogenic Escherichia coli; prevent; 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）：快速检测食品和水传播病原体的能力对于预防与我国食品和水供应污染相关的疫情至关重要。现有的检测系统同时筛选单个样本中多种物质的能力有限。为了满足这一需求，我们建议将连接酶检测反应 (LDR) 与 PCR 和通用阵列检测相结合，我们已经在识别和区分血源性细菌和病毒病原体（包括 A 类生物威胁制剂）方面进行了验证。我们将把这些测定转移到 Cepheid GeneXpert 系统上，并评估它们在模块化微流体装置中的性能。上述技术将用于满足本应用程序的特定目标： 目标 1. 从单个样品中同时鉴定多种食品和水生细菌。我们将开发多重和巢式 PCR/LDR 检测来鉴定 B 类细菌病原体（空肠弯曲杆菌、小肠结肠炎耶尔森菌、沙门氏菌、志贺氏菌、腹泻性大肠杆菌、致病性弧菌和单核细胞增生李斯特氏菌）。腹泻病患者的粪便标本将在微生物学实验室进行评估，并使用上述分子技术进行测试。随机样本中还将掺入不常见细菌病原体的 DNA，以模拟这些病原体的感染。 目标 2. 从单个样品中同时鉴定多种食品和水生原生动物和病毒。 PCR/LDR和RT-PCR/LDR将分别用于检测B类食品和水源性原虫（小隐孢子虫、卡亚坦环孢子虫、溶组织内阿米巴、贾第鞭毛虫和微孢子虫）和病毒（杯状病毒、甲型肝炎病毒和轮状病毒）。疑似原虫或病毒感染患者的粪便标本将在微生物实验室进行评估，并使用这些分子技术进行测试。 目标 3. 开发使用分子特征谱筛选多种食品和水源病原体的高通量方法。将使用目标 1 和 2 中开发的技术建立使用通用液体处理机器人平台的标准化方案。该测试将被验证用于从 3,000 个粪便样本中检测细菌病原体、原生动物和病毒。 PCR/LDR 检测将迁移至 Cepheid GeneXpert 系统以及其他微流体装置，最初使用毛细管电泳或 LDR-FRET，随后使用通用阵列读数。