Multi-lab validation of a real-time PCR assay for detection and surveillance of SARS-CoV-2 for companion animal applications.

项目摘要 自2019年出现以来，SARS-CoV-2在全球蔓延，导致超过7.57亿例确诊病例。 新冠肺炎和全球680多万人死亡(世卫组织COVID19仪表板，截至2023年2月27日)。的变种 在整个大流行期间都出现了关注(VOC)(疾控中心，2021年)。其中最近的一家是达美航空， 与致病性增强有关，以及奥米克隆及其目前流行的亚型， 这与增加的传播率有关(CDC，2021A)。尽管人类一直是 除了SARS-CoV-2的主要宿主外，动物中也有自然感染的报道(Singla等人，2020年)， 主要是伴侣动物，包括猫和狗，或其他在人类照顾下的动物，包括 老虎、狮子、大猩猩、雪豹、水獭和斑点鬣狗(美国农业部-APHIS)。从2021年8月到现在， 美国所有确诊的伴生和外来物种SARS-CoV-2病例都与 Delta或Omicron，它们也继续代表着这些动物总体感染的大多数 大流行开始以来的人口数量(美国农业部-APHIS)。准确地检测和区分 主要挥发性有机化合物对监测这些动物种群中的SARS-CoV-2很重要，并可作为 回溯性研究的有用工具，可能有助于确定这些物种在 大流行。我们之前已经建立了一种用于检测SARS的多重实时聚合酶链式反应(QPCR)方法。 CoV-2与Delta和Omicron变异体的分化(Tsui等人，2022)。我们的目标是执行一个多- 对当前化验或更新的化验进行实验室验证，并将该化验公开提供给 当前和大流行时代动物提交的SARS-CoV-2监测和诊断研究， 分别进行了分析。具体提案目标如下： 1.对SARS-CoV-2基因组序列进行电子重新分析，以确保检测到当前 循环中的欧米克龙亚变异体，同时仍保持对Delta变异体的检测和区分。 2.通过制作SARS-CoV-2阴性犬鼻拭子，开发用于多实验室验证的样本板 将以不同浓度添加SARS-CoV-2阳性动物样本的池基质。 只有在没有阳性动物样本的情况下，才会使用合成的靶向RNA。绝对的 每个稀释液的浓度将通过数字聚合酶链式反应进行测量，并与定量聚合酶链式反应检测进行比较。 3.将验证样本板分发给协作实验室进行多实验室验证。测试 评审团的材料将包括核糖核酸提取试剂、引物、探针、qPCR反应试剂。 和积极的对照。 4.将收集来自每个协作实验室的实时聚合酶链式反应数据，并进行分析以供实验室间使用 表演，然后总结为会议演示并起草成手稿供 出版。

Project Summary Following its emergence in 2019, SARS-CoV-2 spread globally, leading to over 757 million confirmed cases of COVID-19 and over 6.8 million deaths worldwide (WHO COVID19 dashboard, as of Feb 27, 2023). Variants of concern (VOC) have emerged throughout the pandemic (CDC, 2021a). Among the recent of these are Delta, which has been associated with increased pathogenicity, and Omicron and its currently circulating subtypes, which are associated with increased transmissibility (CDC, 2021a). Although humans have served as the primary SARS-CoV-2 reservoir, natural infections have also been reported in animals (Singla et al., 2020), mostly either companion animals, including cats and dogs, or other animals under human care, including tigers, lions, gorillas, snow leopards, otters and spotted hyenas (USDA-APHIS). From August 2021 to present, all confirmed cases of SARS-CoV-2 in companion and exotics species in the U.S have been associated with either Delta or Omicron, which also continue to represent a majority of overall infections in these animal populations since the beginning of the pandemic (USDA-APHIS). Accurate detection and differentiation of major VOCs is important for surveillance of SARS-CoV-2 in these animal populations, and can serve as a useful tool for retrospective studies that may help determine the roles these species played during the pandemic. We have previously developed a multiplex real-time PCR (qPCR) assay for the detection of SARS- CoV-2 and differentiation of Delta and Omicron variants (Tsui et al., 2022). Our objective is to perform a multi- lab validation of the current assay, or an updated assay, and to make the assay publicly available for surveillance and diagnostic research of SARS-CoV-2 in current and pandemic-era animal submissions, respectively. Specific proposal objectives are as follows: 1. In silico re-analysis of SARS-CoV-2 genome sequences to ensure the assay is detecting the currently circulating Omicron sub-variants while still maintaining detection and differentiation of Delta variant. 2. Develop a sample panel for multi-lab validation by creating a SARS-CoV-2-negative canine nasal swab pool matrix that will be spiked, at different concentrations, with SARS-CoV-2 positive animal samples. Synthesized target RNA will be used only if positive animal samples are not available. Absolute concentration of each dilution will be measured by digital PCR, and compared to qPCR testing. 3. Distribute the validation sample panel to collaborating laboratories for multi-lab validations. Testing materials for the panel will include RNA extraction reagents, primers, probes, qPCR reaction reagents and positive controls. 4. Real-time PCR data from each collaborative laboratory will be collected, analyzed for inter-lab performance, then summarized for conference presentation and drafted into a manuscript for publication.