建立一步法 RT-PCR 检测方法，用于检测猪和禽粪便样本中的泛冠状病毒（α-、β-、γ-和 δ-冠状病毒），使用新设计的猪和禽粪便样本的简并引物。
Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples.
机构信息
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, 44691, United States; College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, 44691, United States.
出版信息
J Virol Methods. 2018 Jun;256:116-122. doi: 10.1016/j.jviromet.2018.02.021. Epub 2018 Feb 27.
Coronaviruses (CoVs) are critical human and animal pathogens because of their potential to cause severe epidemics of respiratory or enteric diseases. In pigs, the newly emerged porcine deltacoronavirus (PDCoV) and re-emerged porcine epidemic diarrhea virus (PEDV) reported in the US and Asia, as well as the discovery of novel CoVs in wild bats or birds, has necessitated development of improved detection and control measures for these CoVs. Because the previous pancoronavirus (panCoV) RT-PCR established in our laboratory in 2007-2011 did not detect deltacoronaviruses (δ-CoVs) in swine fecal and serum samples, our goal was to develop a new panCoV RT-PCR assay to detect known human and animal CoVs, including δ-CoVs. In this study, we designed a new primer set to amplify a 668 bp-region within the RNA-dependent RNA polymerase (RdRP) gene that encodes the most conserved protein domain of α-, β-, γ-, and δ-CoVs. We established a one-step panCoV RT-PCR assay and standardized the assay conditions. The newly established panCoV RT-PCR assay was demonstrated to have a high sensitivity and specificity. Using a panel of 60 swine biological samples (feces, intestinal contents, and sera) characterized by PEDV, PDCoV and transmissible gastroenteritis virus-specific RT-PCR assays, we demonstrated that sensitivity and specificity of the newly established panCoV RT-PCR assay were 100%. 400 avian fecal (RNA) samples were further tested simultaneously for CoV by the new panCoV RT-PCR and a one-step RT-PCR assay with the δ-CoV nucleocapsid-specific universal primers. Four of 400 avian samples were positive for CoV, three of which were positive for δ-CoV by the conventional RT-PCR. PanCoV RT-PCR fragments for 3 of the 4 CoVs were sequenced. Phylogenetic analysis revealed the presence of one γ-CoV and two δ-CoV in the sequenced samples. The newly designed panCoV RT-PCR assay should be useful for the detection of currently known CoVs in animal biological samples.
冠状病毒（CoVs）是重要的人类和动物病原体，因为它们有可能引起严重的呼吸道或肠道疾病流行。在美国和亚洲，新出现的猪德尔塔冠状病毒（PDCoV）和重新出现的猪流行性腹泻病毒（PEDV），以及在野生蝙蝠或鸟类中发现的新型 CoVs，都需要开发改进的检测和控制这些 CoVs 的措施。由于我们实验室在 2007-2011 年建立的先前的泛冠状病毒（panCoV）RT-PCR 未能在猪粪便和血清样本中检测到德尔塔冠状病毒（δ-CoVs），我们的目标是开发一种新的 panCoV RT-PCR 检测方法，以检测已知的人类和动物 CoVs，包括 δ-CoVs。在这项研究中，我们设计了一组新的引物，以扩增 RNA 依赖性 RNA 聚合酶（RdRP）基因内的 668bp 区域，该基因编码 α-、β-、γ-和 δ-CoVs 最保守的蛋白结构域。我们建立了一步 panCoV RT-PCR 检测方法，并对检测条件进行了标准化。新建立的 panCoV RT-PCR 检测方法具有很高的灵敏度和特异性。使用一组 60 个猪生物样本（粪便、肠内容物和血清），这些样本通过 PEDV、PDCoV 和传染性胃肠炎病毒的特异性 RT-PCR 检测进行了特征分析，我们证明了新建立的 panCoV RT-PCR 检测方法的灵敏度和特异性均为 100%。同时，用新的 panCoV RT-PCR 和一步 RT-PCR 检测试剂盒对 400 个禽粪便（RNA）样本进行了 CoV 检测，该试剂盒使用了δ-CoV 核衣壳特异性通用引物。在 400 个禽样中有 4 个 CoV 阳性，其中 3 个通过常规 RT-PCR 检测为δ-CoV 阳性。对 4 个 CoV 中的 3 个进行了 panCoV RT-PCR 片段测序。系统发育分析显示，测序样本中存在一种γ-CoV 和两种δ-CoV。新设计的 panCoV RT-PCR 检测方法应可用于检测动物生物样本中目前已知的 CoVs。
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