Development of multiplex real-time PCR assays for differentiating SARS-CoV-2 from other respiratory and enteric pathogens

项目总结/摘要 迫切需要开发和验证新的和改进的分子和生物技术。 用于伴侣动物中SARS-CoV-2诊断的血清学试验（即，狗和猫）。它 研究表明，COVID-19患者经常将SARS-CoV-2传染给他们的宠物。猫 比狗更容易感染SARS-CoV-2，有证据表明这两种物种都可以 在自然或实验性暴露后患上COVID-19。2019冠状病毒病的临床症状 猫和狗包括呼吸道症状和腹泻，这必须与猫科动物区分开来。 呼吸道疾病综合征（FRDC）、犬传染性呼吸道疾病综合征（CIRDC或 通常称为"犬舍咳嗽"），以及由其他病毒、细菌和 寄生虫因此，SARS-CoV-2的快速检测和与其他常见病毒的鉴别， 单一标本中的细菌因子对于控制COVID-19和实施 适当的公共卫生措施。同样，迫切需要高度发展 特异性和灵敏性抗体检测（血清学）分析，以回答基本问题 关于SARS-CoV-2的流行病学和免疫反应，并协助实施， 控制方案的评估。因此，假设目前的COVID-19实验室检测 通过开发新的多重实时qPCR/RT-qPCR可以改善犬和猫中的19 用于检测和区分呼吸道（鼻和 鼻咽）和粪便标本，以及开发用于检测的夹心ELISA 的IgM和IgG免疫应答。为了测试这个中央 假设，我们有两个具体目标：目标1）。开发多重实时PCR检测试剂盒， 检测SARS-CoV-2和其他犬猫呼吸道和肠道病原体： 目的是开发一系列多重（四重）TaqMan®实时qPCR/RT-qPCR 使用荧光标记的探针的组合进行测定。将使用以下方法优化所有测定 新的TaqPath 1-Step Multiplex Master Mix;和Aim 2）。夹心ELISA的研制 犬、猫血清SARS-CoV-2抗体检测：IgM、IgG检测 使用重组核衣壳（N）和 刺突（S）蛋白。总之，多重实时qPCR/RT-qPCR检测具有高度灵敏度 并且具有特异性，可以快速识别和区分感染了SARS-CoV-2的猫和狗。 IgM和IgG ELISA的开发和验证将允许识别暴露的动物 对病毒的反应这些新开发的检测方法将提高分子和血清学诊断 宠物中的SARS-CoV-2，以及先进的流行病学调查。

PROJECT SUMMARY/ABSTRACT There is an urgent need for developing and validating new and improved molecular and serological tests for the diagnosis of SARS-CoV-2 in companion animals (i.e., dogs and cats). It has been shown that people with COVID-19 frequently pass SARS-CoV-2 on to their pets. Cats are more susceptible to SARS-CoV-2 than dogs, and there is evidence that both species can develop COVID-19 after a natural or experimental exposure. The clinical signs of COVID-19 in cats and dogs include respiratory signs and diarrhea, which must be differentiated from feline respiratory disease complex (FRDC), canine infectious respiratory disease complex (CIRDC or commonly known as “kennel cough”), and diarrhea caused by other viruses, bacterial and parasites. Thus, rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents in a single specimen is critical in controlling COVID-19 and implementing appropriate public health measures. Similarly, there is an urgent need for developing highly specific and sensitive antibody detection (serological) assays to answer fundamental questions about the epidemiology and immune response to SARS-CoV-2 and aid in the implementation and evaluation of control programs. Thus, it is hypothesized that current laboratory testing for COVID- 19 in dogs and cats could be improved by developing new multiplex real-time qPCR/RT-qPCR assays for the detection and differentiation of SARS-CoV-2 in respiratory (nasal and nasopharyngeal) and fecal specimens, as well as developing a sandwich ELISA for the detection of IgM and IgG immune responses using recombinant viral proteins. To test this central hypothesis, we have two specific aims: Aim 1). Develop Multiplex Real-time PCR Assays to Detect SARS-CoV-2 and Other Respiratory and Enteric Pathogens in Dogs and Cats: The objective is to develop a series of multiplex (quadruplex) TaqMan® real-time qPCR/RT-qPCR assays using a combination of fluorescently labeled probes. All assays will be optimized using the new TaqPath 1-Step Multiplex Master Mix; and Aim 2). Develop Sandwich ELISA for the Detection of Antibodies to SARS-CoV-2 in Dog and Cat Serum Samples: To detect IgM and IgG immune response to SARS-CoV-2 in dogs and cats using recombinant nucleocapsid (N) and spike (S) proteins. In summary, multiplex real-time qPCR/RT-qPCR assays are highly sensitive and specific and will rapidly identify and distinguish dogs and cats infected with SARS-CoV-2. The development and validation of both IgM and IgG ELISA will allow identifying animals exposed to the virus. These newly developed assays will enhance the molecular and serologic diagnosis of SARS-CoV-2 in pets, and also, advance epidemiological investigations.