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

项目摘要 美国食品和药物管理局(FDA)监管宠物食品，要求其食用安全，含有 没有有害物质，是在卫生条件下产生的，并真实贴上标签。 然而，这些食品不需要上市前的批准，因为它们是用配料制成的 被认为是安全的。宠物食品被证明在加工过程中会在植物中掺假， 导致动物和人类与食源性病原体有关的疾病。植物加工 也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 导致宠物食品含有意想不到的成分，导致虚假标签。是否故意 不管是不是，在宠物食品中掺入来自未标记属的DNA的情况此前都曾被检测到。 目前用于检测食源性病原体的方法是劳动密集型的，可能需要几个 几天的时间来获得可行的结果。此外，样本很可能要送到几个 用于全面测试(例如，病毒、细菌和寄生虫)和检测的不同实验室 属于食物中的肉源的属。其他测试功能，包括 快速检测和初步表征的综合方法将有益于 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)小组，它包括 与食源性疾病相关的多种病原体的检测和初始检测用引物 对涉及的病原体(S)进行表征，并对 产品。在进行多重聚合酶链式反应之前，可以在NGS工作流程中加入多个引物 测序。使用引子可以使NGS协议标准化，以实现一致性 跨多个实验室的性能，并通过以下方式简化技术的使用和实施 那些已经熟悉了聚合酶链式反应和桑格测序的人。这导致了NGS的实施 在诊断实验室环境中相对简单，即使NGS生物信息管道仍然 需要进一步开发和验证实验室间的标准化。简单化的分析 从目标NGS面板生成的数据减少了这一限制。此外，作为一项 结果在最初的聚合酶链式反应扩增步骤中，可以直接从临床进行检测 取样或跟随浓缩过程。所有这些优势表明，有针对性的NGS 可用于食源性农药残留的全面检测 具有最初病原体特征和肉源属检测的病原体，具有 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.