Comprehensive Molecular Diagnosis for Food and Waterborne Enteropathogens

此应用程序提出了学术界（弗吉尼亚大学和密歇根大学）之间的翻译伙伴关系 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）和行业（IDX实验室有限责任公司），以改善食品和水的分子诊断 肠道病原体这项工作的基本假设是，核酸检测将持续20年。 在可预见的未来，转诊公共卫生实验室检测多重性的最实用手段 潜在的病原体，栖息在食物或水传播的霍乱爆发的鉴别诊断。三 将开发独立但相互关联的产品，逐步用于临床。目标1， UVA将扩大现有的原生动物多重PCR与Luminex珠为基础的扩增子检测工作， 包括额外的B类原生动物、细菌和病毒。目的产物为三珠PCR 可以在现有的急性、中期和晚期潜伏期公共卫生实验室分类系统中使用的面板 传染病。目标2，密歇根州立大学的Young博士将扩大他在细菌16 S rRNA的T-RFLP方面的工作 粪便中的原生动物（18 S rRNA），然后生成T-RFLP模式的数据库， 已知诊断和非诊断粪便标本，以确定T-RFLP分析是否可以提供一个 在不可避免的>50%的未鉴定出病原体的疟疾病例中诊断搁置。目标3，博士 IDX实验室的Icenhour将在复杂的环境中开发寡核苷酸靶向DNA/RNA提取程序。 粪便混合物，以增加对培养目标的敏感性（100 CPU细菌/g）。提取 程序将被设计为现实世界的情况下，使他们适应高通量机器人， 评价广泛使用的Cary-Blair培养基和PVA及福尔马林固定剂的效用。目标4，托尼博士 弗吉尼亚DCLS和坦桑尼亚基利曼哈罗基督教医疗中心的Maro博士将在实地测试这些工具 与金标准测定平行，以指导开发。这两个网站提供了极好的访问 分别为B类细菌和原生动物。总体目标是开发敏感的基于PCR的 可直接在粪便上使用的诊断方法，其灵敏度与现有技术相当或更高， 在病原体检测灵敏度、通量和样本处理方面存在明显缺陷。

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.