GD Biotech, 3595 Route de Tournai, 59501 Douai, France.
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
J Immunol Methods. 2020 Aug;483:112808. doi: 10.1016/j.jim.2020.112808. Epub 2020 Jun 17.
Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.
三种不同的猪肠道冠状病毒（PECs），即猪流行性腹泻病毒（PEDV）、传染性胃肠炎病毒（TGEV）和猪德尔塔冠状病毒（PDCoV），目前在美国商业猪群中流行。PECs 的鉴别诊断依赖于实验室方法。本研究描述了一种基于病毒特异性重组 S1 蛋白的 ELISA 样多重平面免疫分析的开发，该蛋白以阵列形式点状印刷在 96 孔微量滴定板的底部，用于同时检测单个样品中 PEDV、TGEV 和 PDCoV 的差异血清学诊断。该技术的总体格式和工作原理与固相标准 ELISA 相似。在三个典型的孵育步骤之后，通过观察蓝色斑点的显色情况来显示反应结果，其强度与阵列中针对特定病毒抗原靶标的抗体水平相关。该检测方法的诊断性能通过在实验条件下用 PEDV、TGEV Purdue、TGEV Miller、PDCoV（美国/IL/2014）或培养基接种的猪在不同时间（接种后第 7、0、7、14、21、28、35 和 42 天）采集的已知状态血清样本（n=480）进行评估。为了确保针对每个给定抗原靶标具有 100%的诊断和分析特异性，选择了抗原特异性截断值。PEDV S1 的总体诊断敏感性为 92%（44/48 阳性，95%置信区间[CI]98,100），TGEV S1 为 100%（95/95 阳性，95%CI 98,100），PDCoV S1 为 98%（47/48 阳性，95%CI 97,100）。本研究结果表明，AgroDiag PEC 多重免疫分析是一种用于 PEDV、TGEV 和 PDCoV 差异检测和血清学诊断的高效可靠的检测方法。
