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.

目前，美国商业猪群中正在流行三种不同的猪肠道冠状病毒（PEC），即猪流行性腹泻病毒（PEDV），传染性胃肠炎病毒（TGEV）和猪三角冠状病毒（PDCoV）。PEC的鉴别诊断依赖于实验室方法。这项研究描述了一种基于ELISA的多重平面免疫测定方法的开发，该方法基于印在96孔微孔板底部斑点阵列中的病毒特异性重组S1蛋白，可同时检测PEDV，TGEV，PDCoV的血清学差异样品。技术的总体格式和工作原理类似于固相标准ELISA。在三个典型的孵化步骤之后，反应显示为蓝点，其强度与阵列中针对特定病毒抗原靶标的抗体水平相关。对已知状态的血清样品进行了测定的诊断性能（n  = 480）随时间（接种后第7、0、7、14、21、28、35和42天）从接种PEDV，TGEV Purdue，TGEV Miller，PDCoV的猪中收集（美国/ IL / 2014） ，或在实验条件下用培养基接种的模拟物。选择抗原特异性的临界值以确保对每个给定的抗原靶标具有100％的诊断和分析特异性。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的总体诊断敏感性为100％（95/95阳性，95％CI 98，100），98％（47 / PDCoV S1的/ 48阳性，95％CI 97，100）。这项研究的结果表明，AgroDiag PEC多重免疫测定法是对PEDV，TGEV和PDCoV进行差异检测和血清诊断的有效且可靠的测试方法。