ABSTRACT

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 10² copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

摘要

随着2019年新型严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的爆发，冠状病毒已成为全球病毒学领域的研究热点。冠状病毒主要引起呼吸道和消化道疾病，多种冠状病毒可引起猪腹泻，如猪流行性腹泻病毒（PEDV）、猪三角洲冠状病毒（PDCoV）和新发猪急性腹泻综合征冠状病毒（SADS-CoV）。这些病毒造成了巨大的经济损失，并被认为是潜在的公共卫生威胁。猪环病毒（PToV）与冠状病毒具有相似的基因组结构和复制策略，同属于巢病毒目。在这里，我们首次开发了一种基于多重Taq Man 探针的实时 PCR，用于同时检测 PEDV、PDCoV、PToV 和 SADS-CoV。针对 PDEV、PToV 和 SADS-CoV 的 ORF1a 区域以及 PDCoV 的 ORF1b 区域设计了特异性引物和Taq Man 荧光探针。该方法具有较高的灵敏度和特异性，每种病原体的检测限为1 × 10 ^{2 个拷贝/μL。}采用该方法对101份有腹泻症状的临床猪样本进行了分析，结果与常规逆转录PCR（RT-PCR）结果具有良好的一致性。该方法提高了对这些新出现和重新出现的猪肠道病毒的监测效率，有助于减少养猪业的经济损失，这也有利于动物和公众健康。