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供水污染相关的疫情至关重要。现有的检测系统具有有限的能力来同时筛选单个样品的多种试剂。为了满足这一需求，我们建议使用连接酶检测反应（LDR）结合PCR和通用阵列检测，我们已经验证了在识别和区分血液传播的细菌和病毒病原体，包括A类生物威胁剂。我们将把这些检测转移到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申请的特定目标： 目标1。从单一样品中同时鉴定多种食物和水传播的细菌。我们将开发多重和巢式PCR/LDR检测方法来鉴定B类细菌病原体（空肠弯曲杆菌、小肠结肠炎耶尔森氏菌、沙门氏菌、志贺氏菌、腹泻性大肠杆菌）。大肠杆菌、致病性弧菌属和单核细胞增生李斯特菌）。将在微生物学实验室评价来自腹泻病患者的粪便标本，并使用上述分子技术进行检测。随机样本还将加标不常见细菌病原体的DNA，以模拟这些病原体的感染。 目标2.从一个样品中同时鉴定多种食物和水源原生动物和病毒。PCR/LDR和RT-PCR/LDR将分别用于检测B类食物和水传播的原生动物（隐孢子虫、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学实验室进行评价，并使用这些分子技术进行检测。 目标3。利用分子特征谱建立高通量的食品和水传播病原体筛查方法。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检测3,000份粪便样本中的细菌病原体、原生动物和病毒。PCR/LDR检测试剂盒将迁移至Cepheid GeneXpert系统以及其他微流体设备，最初使用毛细管电泳或LDR-FRET，随后使用通用阵列读数。

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.