Division of Infectious Diseases, Duke University School of Medicine, Durham, North Carolina, USA; Duke Global Health Institute, Duke University, Durham, North Carolina, USA; NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Division of Infectious Diseases, Duke University School of Medicine, Durham, North Carolina, USA; Duke Global Health Institute, Duke University, Durham, North Carolina, USA.
J Clin Virol. 2020 Jul;128:104391. doi: 10.1016/j.jcv.2020.104391. Epub 2020 Apr 30.
During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats.
To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily.
We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 10 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types.
This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.
在过去的二十年中，三种新型冠状病毒（CoV）已出现并引发国际人类传染病，导致严重的发病率。CoV 也已在动物中出现并引发严重的传染病。为了帮助减轻这些威胁，需要更好地了解 CoV 的自然宿主和遗传多样性。
设计并评估一种分子诊断工具，用于检测和鉴定所有目前已识别和潜在未来出现的 Orthocoronavirinae 亚科 CoV。
我们基于 38 个人类和动物 CoV 的已发表基因组序列设计了一种半嵌套、逆转录 RT-PCR 检测方法。我们用 14 种人和动物 CoV 以及 11 种其他非 CoV 呼吸道病毒评估了该检测方法。通过对检测方法的靶标扩增子进行测序，该检测方法正确识别了每种 CoV；未观察到与 11 种常见呼吸道病毒的交叉反应。检测限取决于所测试的 CoV 种类，范围从 4 到 4×10 拷贝/反应。为了评估该检测方法的临床性能，我们测试了一个之前研究的大样本组：192 份来自肺炎患者的人呼吸道样本、5 份 COVID-19 患者的临床样本、81 份禽类口腔分泌物样本、109 份猪粪浆样本和 31 份活禽市场气溶胶样本。所有 RT-PCR 阳性样本的扩增子均通过 Sanger 测序确认。我们的检测方法在所有测试样本和所有样本类型中表现良好。
该检测方法可用于检测和鉴定所有以前识别的 CoV，包括 SARS-CoV-2 以及 Orthocoronavirinae 亚科中任何潜在出现的 CoV。
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