Comprehensive next-generation sequencing panel for foodborne pathogen detection and meat speciation

项目摘要 食品和药物管理局（FDA）管理宠物食品，要求它可以安全食用， 无有害物质，使用卫生条件生产，并如实标示。 然而，这些食品不需要上市前批准，因为它们是用配料制成的 认为安全。宠物食品掺假已被证明发生在植物加工过程中， 导致动物和人类与食源性病原体相关的疾病。植物加工 还可能导致用于宠物食品生产的产品之间的交叉污染。这可以 导致宠物食品含有非预期成分，导致虚假标签。无论是有意 或者不掺杂，以前已经检测到宠物食品中掺杂了来自未标记属的DNA。 目前用于检测食源性病原体的方法是劳动密集型的， 天，以获得可操作的结果。此外，样本可能必须发送到几个 不同的实验室进行全面测试（例如病毒、细菌和寄生虫）和检测 食物中肉类来源的种类。其他测试功能，包括 快速检测和初步表征的综合方法，将有利于 快速召回掺假食品。 我们建议开发一个有针对性的下一代测序（NGS）面板，其中包括 与食源性疾病相关的多种病原体的引物，用于检测和初始 所涉及的病原体的表征，以及用于表征 产品引物可以在扩增之前并入NGS工作流程中用于多重PCR。 测序引物的使用允许NGS方案的标准化，以获得一致的结果。 通过以下方式简化技术的使用和实施 那些已经熟悉PCR和桑格测序的人。这使得NGS的实施 在诊断实验室环境中相对简单，尽管NGS生物信息学管道仍然 需要进一步开发和验证，以实现跨实验室的标准化。简单的分析 从目标NGS面板生成的数据的有效性减轻了这种限制。此外，作为 根据初始PCR扩增步骤的结果，该测试可以直接从临床上进行。 样品或富集过程之后。所有这些优势都表明，有针对性的NGS 可用于提供检测食源性 病原体与最初的病原体特征和检测肉源属， 比目前使用的方法更快的周转时间。

Project Summary The Food and Drug Administration (FDA) regulates pet food, requiring it is safe to eat, contains no harmful substances, is produced using sanitary conditions, and is truthfully labeled. However, the foods do not need pre-market approval because they are made with ingredients considered safe. Pet food adulteration has been shown to occur in plants during processing, resulting in illness in animals and humans related to foodborne pathogens. Plant processing may also result in cross-contamination between products used for pet food production. This can result in pet foods having unintended ingredients, leading to false labelling. Whether intentional or not, adulteration of pet food with DNA from unlabeled genera has been previously detected. Current methods used to detect foodborne pathogens are labor-intensive and may take several days to obtain actionable results. Additionally, samples likely have to been sent to several different labs for comprehensive testing (e.g. for virus, bacteria, and parasites) and for detection of the genus of the meat source in the food. Additional testing capabilities, including comprehensive methods for rapid detection and initial characterization, would be beneficial for rapid recall of adulterated food. We propose to develop a targeted next-generation sequencing (NGS) panel, which incorporates primers for multiple pathogens associated with foodborne illness, for detection and initial characterization of the involved pathogen(s), and for characterization of the meat included in the product. Primers can be incorporated into the NGS workflow for multiplex PCR prior to sequencing. Use of primers allows for standardization of the NGS protocol for consistent performance across multiple labs and eases the use and implementation of the technology by those already familiar with PCR and Sanger sequencing. This renders implementation of NGS relatively simple in the diagnostic lab setting, even though NGS bioinformatics pipelines still need further development and validation for standardization across labs. The simplistic analysis of the data generated from a targeted NGS panel mitigates this limitation. Additionally, as a result of the initial PCR amplification steps, the test can be performed directly from the clinical sample or following an enrichment process. All of these advantages suggest that targeted NGS can be used to provide a comprehensive diagnostic assay for the detection of foodborne pathogens with initial pathogen characterization and detection of meat source genera, that has a more rapid turn-around time than currently used methods.