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， COVID-19和全球超过680万例死亡（WHO COVID 19仪表板，截至2023年2月27日）。的变体 在整个大流行期间出现了VOC（VOC）问题（CDC，2021 a）。其中最近的是三角洲， 它与致病性增加有关，而Omicron及其目前流行的亚型， 这与增加的传播性有关（CDC，2021 a）。虽然人类已经成为 原发性SARS-CoV-2储库，也报道了动物中的自然感染（Singla等，2020年）， 大多数是伴侣动物，包括猫和狗，或其他受人类照顾的动物，包括 老虎、狮子、大猩猩、雪豹、水獭和斑点鬣狗（USDA-APHIS）。2021年8月至今， 美国所有确诊的伴侣和外来物种中的SARS-CoV-2病例都与以下疾病有关： Delta或Omicron，它们也继续代表这些动物中大部分总体感染 （USDA-APHIS）。准确检测和区分 主要挥发性有机化合物对于监测这些动物群体中的SARS-CoV-2非常重要，可以作为 回顾性研究的有用工具，可能有助于确定这些物种在 流行病我们之前已经开发了一种用于检测SARS的多重实时PCR（qPCR）检测方法， CoV-2和Delta和Omicron变体的分化（Tsui等人，2022年）。我们的目标是执行一个多- 对当前检测试剂盒或更新后的检测试剂盒进行实验室验证，并使该检测试剂盒可公开用于 在当前和大流行时期提交的动物中进行SARS-CoV-2的监测和诊断研究， 分别具体建议目标如下： 1.对SARS-CoV-2基因组序列进行计算机再分析，以确保检测试剂盒能够检测到当前 循环Omicron亚变体，同时仍然保持Delta变体的检测和区分。 2.通过创建SARS-CoV-2阴性犬鼻拭子，开发用于多实验室验证的样本组 将以不同浓度加标SARS-CoV-2阳性动物样本的混合基质。 只有在没有阳性动物样本时，才使用合成的靶RNA。绝对 将通过数字PCR测量各稀释液的浓度，并与qPCR检测进行比较。 3.将验证样品组分发给合作实验室进行多实验室验证。测试 试剂盒材料包括RNA提取试剂、引物、探针、qPCR反应试剂 和阳性对照。 4.将收集来自每个合作实验室的实时PCR数据，进行实验室间分析 性能，然后总结会议演示文稿，并起草成一份手稿， 出版物

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.