Increasing the diagnostic PCR and next-generation sequencing capacity for the diagnosis and variant detection of COVID-19 in animals at the North Dakota State University Veterinary Diagnostic Lab

北达科他州立大学兽医诊断实验室提高诊断 PCR 和下一代测序能力，用于动物中 COVID-19 的诊断和变异检测

• 批准号: 10449513
• 负责人: Brianna Stenger
• 金额: $ 5.99万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449513
• 关键词: Increasing; diagnostic; PCR; next; generation

Summary The primary goal of this project is to increase the capacity for next-generation sequencing (NGS) and SARS-CoV-2 variant detection for the diagnosis of COVID-19 in animals by validating commercially available reagent kits for extraction, PCR, and NGS using a dedicated one-way NGS workflow. This will add the NDSU-VDL to the network of veterinary diagnostic laboratories capable and proficient at testing and sequencing SARS-CoV- 2 in samples from animals. To build this capacity, reagents and dedicated equipment for a one- way workflow are needed. There are three specific aims. 1) Validate the Applied Biosystems™ COVID-19 TaqPath™ Combo Kit for the detection of SARS-CoV-2 and diagnosis of COVID-19 in animal samples. Targeted samples include tissue homogenates and swabs (nasal, oral, fecal) from animals. Extracted product can be used for downstream next generation sequencing on long or short read platforms, such as the MinION or iSeq, respectively. 2) Create a safe, one- way workflow for next generation sequencing that will minimize contamination and increase sequencing capacity by allowing multiple technicians to work on different steps in the process at the same time. 3) Diagnose SARS-CoV-2 variants through next generation sequencing on the MinION platform. This will be accomplished using the NEBNext® ARTIC SARS-CoV-2 Companion Kit and protocol for the MinION for NGS and following the EPI2ME analysis pipeline. The use of the flongle flow cells will be evaluated to reduce sequencing costs. In addition, the amount of time, cost, and ease of use of this NGS library prep kit and analysis pipline will the compared with other laboratories evaluating different kits.

摘要 该项目的主要目标是增加下一代网络的容量 序列分析和SARS-CoV-2变异株检测在新冠肺炎诊断中的应用 通过验证用于提取、PCR和NGS的商业可用试剂盒来验证动物 使用专用的单向NGS工作流。这会将NDSU-VDL添加到网络 有能力并精通SARS冠状病毒检测和测序的兽医诊断实验室- 2在动物样本中。为了建设这一能力，试剂和专用设备- 需要工作流程的方式。有三个具体目标。1)验证应用生物系统™ 新冠肺炎TaqPath™组合试剂盒用于SARS-CoV-2检测和新冠肺炎诊断 在动物样本中。目标样本包括组织匀浆和拭子(鼻腔、口腔、粪便) 从动物身上。提取的产物可用于下游的下一代测序 长读或短读平台，如minion或iseq。2)创建一个安全的、一个- 下一代测序的工作流程将最大限度地减少污染并增加 通过允许多名技术人员在流程中的不同步骤上工作来提高排序能力 同一时间。3)通过对SARS-CoV-2变异株的下一代测序诊断 奴才站台。这将使用NEBNext®ARTIC SARS-CoV-2来实现 适用于NGS和EPI2ME分析的伙伴套件和协议 输油管道。将对Flong le Flow电池的使用进行评估，以降低测序成本。在……里面 此外，此NGS库准备工具包和分析的时间、成本和易用性 PIPLINE将与评估不同试剂盒的其他实验室进行比较。