African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 10² copies/μL for ASFV and 6.34 × 10¹ copies/μL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018−2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

非洲猪瘟病毒（ASFV）、经典猪瘟病毒（CSFV）和非典型猪瘟病毒（APPV）给全球养猪业造成了相当大的经济损失，早期准确诊断这些病毒对控制疾病至关重要被他们诱导。本研究根据这些病毒的高度保守基因组区域设计了三对特异性引物，并在各种反应条件下建立了针对ASFV、CSFV和APPV的多重逆转录聚合酶链反应（mRT-PCR）检测方法。优化。mRT-PCR 检测包括两个步骤，即逆转录 (RT) 和 mPCR。该检测方法对 ASFV、CSFV 和 APPV 具有高度特异性、敏感性和可重复性，不会与其他猪病原体发生交叉反应。该测定的灵敏度，ASFV 和 6.34 × 10 ¹为²份/μLCSFV 和 APPV 的拷贝数/μL。通过建立的 mRT-PCR 方法分析了 2018-2019 年华南广西省疑似感染仔猪的 384 份临床样本。结果表明，ASFV、CSFV和APPV的阳性率分别为43.75%、13.28%和4.17%，ASFV/CSFV、ASFV/APPV和CSFV/APPV的共感染率为5.47%、1.83%和1.30%。 ， 分别。为了解广西新发现病毒APPV的流行病学特征，随机选取APPV阳性动物的临床样本进行扩增测序，获得4个APPV毒株的完整基因组序列。系统发育分析表明，广西APPV株系具有高度的遗传多样性。