Comprehensive Molecular Diagnosis for Food and Waterborne Enteropathogens

此申请建议在学术界(弗吉尼亚大学和密歇根大学)之间建立翻译合作伙伴关系 州立大学)、公共卫生部门(弗吉尼亚联邦综合实验室分部 服务)和工业(IDX Labs LLC)，以改进食品和水中的分子诊断 肠道病原体。这项工作的基本假设是，核酸检测将在 在可预见的未来，转诊公共卫生实验室检测多发性肺炎最实用的手段 潜在病原体存在于食物或水传播腹泻暴发的鉴别诊断中。三 将在临床使用的道路上开发独立但相互关联的产品。目标1，胡普特博士 UVA将扩展现有的原生动物多重聚合酶链式反应和Luminex基于珠粒的扩增检测工作，以 包括额外的B类原生动物、细菌和病毒。目的产物为三种珠状聚合酶链式反应 可在现有的公共卫生实验室急性、中期和晚期孵化分诊系统中使用的小组 腹泻病。目标2，密歇根州立大学的杨博士将扩大他在细菌16S rRNA T-RFLP方面的工作 粪便中的群落到原生动物(18S RRNA)，然后生成T-RFLP模式数据库 已知诊断和未诊断的粪便样本以确定T-RFLP分析是否可以提供 在未发现病原体的腹泻病例中，50%的病例将不可避免地归类诊断。Aim 3，Dr. IDX实验室的Icenhour将在复合体中开发寡核苷酸靶向DNA/RNA提取程序 混合粪便以提高对培养目标的敏感性(100个CPU细菌/克)。提取 程序将针对真实世界的场景进行设计，使其适应高吞吐量的机器人和 评估广泛使用的Cary-Blair介质、PVA和福尔马林固定剂的实用性。目标4，托尼博士 弗吉尼亚DCLS和坦桑尼亚乞力马扎罗基督教医疗中心的Maro博士将在现场测试这些工具 与黄金标准化验平行进行，以指导发展。这两个网站提供了极好的访问 B类细菌和原生动物。总体目标是开发敏感的基于聚合酶链式反应的 可直接在粪便上使用的诊断方法，具有与现有技术相当或更好的敏感性，这些技术具有 明确病原体检测、吞吐量和标本处理的灵敏度方面的缺陷。

This application proposes a translational partnership between academia (University of Virginia and Michigan State University), the public health sector (Commonwealth of Virginia Division of Consolidated Laboratory Services), and industry (IDX Labs LLC) to improve molecular diagnosis of food and waterborne enteropathogens. The underlying hypothesis to this work is that nucleic acid detection will remain for the foreseeable future the most practical means for referral public health laboratories to detect the multiplicity of potential pathogens that inhabit the differential diagnosis of a food or waterborne diarrheal outbreak. Three independent but linked products will be developed on pathways towards clinical use. Aim 1, Dr. Houpt of UVA will expand existing work on protozoa multiplex PCR with Luminex bead-based amplicon detection to include the additional Category B protozoa, bacteria, and viruses. The goal product are three bead-PCR panels that can be used within the existing public health lab triage system of acute, mid, and late-incubation diarrheal disease. Aim 2, Dr. Young of MSU will expand his work on T-RFLP for bacterial 16S rRNA communities in stool to the protozoa (18S rRNA) and then generate a database of T-RFLP patterns from known-diagnosis and no-diagnosis stool specimens to determine whether T-RFLP profiling can provide a diagnostic pigeonhole in the inevitable >50% of diarrheal cases where no pathogen is identified. Aim 3, Dr. Icenhour of IDX Labs will develop oligo-targeted DNA/RNA extraction procedures amidst the complex mixture of stool to increase sensitivity towards the goal of culture (100 CPU bacteria/g). Extraction procedures will be designed for the real-world scenario, adapting them to high-throughput robotics and evaluating utility in the widely-used Cary-Blair media and PVA and formalin fixatives. Aim 4, Dr. Toney of Virginia DCLS and Dr. Maro of Kilimanjaro Christian Medical Center, Tanzania will test these tools in the field in parallel with gold-standard assays in order to guide development. These two sites provide superb access to Category B bacteria and protozoa, respectively. The overall goal is to develop sensitive PCR-based diagnostics directly usable on stool with comparable or better sensitivity to existing technologies, which have clear deficincies in sensitivity of pathogen detection, throughput, and specimen handling.