Abstract: To address the challenge of differentiating the major pathogens of viral diarrhea in piglets that cause similar clinical symptoms，including porcine epidemic diarrhea virus（PEDV），transmissible gastroenteritis virus（TGEV），and porcine deltacoronavirus（PDCoV），the highly conserved RdRp genes of these three viruses were used as the target. Specific primers suitable for recombinase‐aided amplification（RAA） were designed and screened，and a triplex RAA assay for the simultaneous detection of PEDV，TGEV，and PDCoV was established.The results showed that after reaction at 37 ℃ for 10 min，specific amplification bands of 137，289，170 bp could be detected by agarose gel electrophoresis.The detection limits for PEDV，TGEV，and PDCoV were 8.9，1.1，7.5 copies/μL，respectively，and no cross‐reactivity was observed with porcine reproductive and respiratory syndrome virus（PRRSV），porcine astrovirus（PAstV），or classical swine fever virus（CSFV）.When applied to 93 clinical samples for parallel comparative testing，the overall detection rate was 5.38%.Compared with the results of previously reported singleplex quantitative PCR assays for PEDV，TGEV，and PDCoV，the coincidence rate reached 100%，demonstrating good clinical application reliability. In summary，this method offers an accurate and efficient new detection tool for the rapid field differentiation of porcine enteric coronaviruses.

Key words: Porcine epidemic diarrhea virus, Transmissible gastroenteritis virus, Porcine deltacoronavirus, Recombinase‐aided amplification, Multiplex detection

摘要： 为应对猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）、猪传染性胃肠炎病毒（Transmissible gastroenteritis virus，TGEV）和猪δ冠状病毒（Porcine deltacoronavirus，PDCoV）等临床症状相似的仔猪病毒性腹泻主要病原的鉴别难题，以这3种病毒高度保守的RdRp基因为靶标，分别设计并筛选出适用于重组酶介导等温扩增（Recombinase aided amplification，RAA）的特异性引物，建立了一种可同步检测PEDV、TGEV和PDCoV的三重RAA检测方法。结果表明，该体系在37 ℃恒温条件下反应10 min，经琼脂糖凝胶电泳即可检测到大小分别为137、289、170 bp的特异性扩增条带；对PEDV、TGEV和PDCoV的检测灵敏度分别为8.9、1.1、7.5 copies/μL，且与猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）、猪星状病毒（Porcine astrovirus，PAstV）及猪瘟病毒（Classical swine fever virus，CSFV）无交叉反应。应用该方法对93份临床样本进行平行比对测试，总体检出率为5.38%，与已报道的PEDV、TGEV、PDCoV单重定量PCR检测结果相比，符合率达100%，显示出良好的临床应用可靠性。综上，该方法为猪肠道冠状病毒的现场快速鉴别提供了一种精准、高效的新型检测工具。

关键词: 猪流行性腹泻病毒, 猪传染性胃肠炎病毒, 猪δ冠状病毒, 重组酶介导等温扩增, 多重检测