Multiplexed Detection of Food and Waterborne Pathogens

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我们国家的食物和水供应污染有关的爆发至关重要。现有的检测系统同时筛选单一样品中多种试剂的能力有限。为了满足这一需求，我们建议使用连接酶检测反应（LDR）结合PCR和通用阵列检测，我们已经在识别和区分血源性细菌和病毒病原体，包括A类生物威胁剂方面进行了验证。我们将把这些检测转移到造父变星GeneXpert系统上，并在模块化微流体装置中评估它们的性能。上述技术将用于满足本应用程序的具体目标：

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.