Multiplexed Detection of Food and Waterborne Pathogens

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防止与我国食品和水供应污染有关的疫情至关重要。现有的检测系统同时筛查单个样本以检测多个试剂的能力有限。为了满足这一需求，我们建议使用连接酶检测反应(LDR)与聚合酶链式反应(PCR)和通用阵列检测相结合，我们已经在鉴定和区分血液传播的细菌和病毒病原体，包括A类生物制剂方面进行了验证。我们将把这些分析转移到Cepheid GeneXpert系统上，并评估它们在模块化微流控设备中的性能。以上技术将用于满足本应用程序的特定目标： 目的1.从单个样品中同时鉴定多种食源性和水源性细菌。我们将发展多重和套式PCR/LDR方法来鉴定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、致病性大肠杆菌、致病性弧菌和单核细胞增生性李斯特菌)。腹泻患者的粪便样本将在微生物实验室进行评估，并使用上述分子技术进行测试。随机样本还将被加入罕见细菌病原体的DNA，以模拟这些病原体的感染。 目的2.从单个样本中同时鉴定多个食物和水传播的原虫和病毒。B类食物和水生原虫(微小隐孢子虫、环孢子虫、溶组织内阿米巴、蓝氏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)将分别被用来检测。疑似原生动物或病毒感染患者的粪便样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。 目的3.建立利用分子特征图谱高通量筛选多种食品和水传播致病菌的方法。将使用AIMS 1和AIMS 2中开发的技术建立使用普通液体处理机器人平台的标准化方案。该测试将用于从3,000个粪便样本中检测细菌病原体、原生动物和病毒。PCR/LDR分析将被移植到Cepheid GeneXpert系统以及其他微流控设备上，首先使用毛细管电泳法或LDR-FRET，然后使用通用阵列读出。

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.