Werid Gebremeskel Mamu, Zhang He, Ibrahim Yassein M, Pan Yu, Zhang Lin, Xu Yunfei, Zhang Wenli, Wang Wei, Chen Hongyan, Fu Lizhi, Wang Yue
Heilongjiang Provincial Key Laboratory of Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Chongqing Academy of Animal Science, Chongqing 408599, China.
Vet Sci. 2022 Apr 7;9(4):176. doi: 10.3390/vetsci9040176.
Swine viruses like porcine sapovirus (SaV), porcine encephalomyocarditis virus (EMCV), porcine rotavirus A (RVA) and porcine astroviruses (AstV) are potentially zoonotic viruses or suspected of potential zoonosis. These viruses have been detected in pigs with or without clinical signs and often occur as coinfections. Despite the potential public health risks, no assay for detecting them all at once has been developed. Hence, in this study, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of SaV, EMCV, RVA and AstV from swine fecal samples. The PCR parameters were optimized using specific primers for each target virus. The assay's sensitivity, specificity, reproducibility, and application to field samples have been evaluated. Using a pool of plasmids containing the respective viral target fragments as a template, the developed mRT-PCR successfully detected 2.5 × 10 copies of each target virus. The assay's specificity was tested using six other swine viruses as a template and did not show any cross-reactivity. A total of 280 field samples were tested with the developed mRT-PCR assay. Positive rates for SaV, EMCV, RVA, and AstV were found to be 24.6% (69/280), 5% (14/280), 4.3% (12/280), and 17.5% (49/280), respectively. Compared to performing separate assays for each virus, this mRT-PCR assay is a simple, rapid, and cost-effective method for detecting mixed or single infections of SaV, EMCV, RVA, and AstV.
猪源病毒，如猪萨波病毒（SaV）、猪脑心肌炎病毒（EMCV）、猪轮状病毒A（RVA）和猪星状病毒（AstV），是潜在的人畜共患病毒或疑似具有潜在人畜共患性。这些病毒在有或没有临床症状的猪中均有检出，且常以混合感染的形式出现。尽管存在潜在的公共卫生风险，但尚未开发出能一次性检测所有这些病毒的检测方法。因此，在本研究中，开发了一种多重逆转录聚合酶链反应（mRT-PCR）检测方法，用于同时从猪粪便样本中检测SaV、EMCV、RVA和AstV。使用针对每种目标病毒的特异性引物对PCR参数进行了优化。对该检测方法的灵敏度、特异性、重复性以及在现场样本中的应用进行了评估。以含有各自病毒目标片段的质粒混合物作为模板，所开发的mRT-PCR成功检测到每种目标病毒2.5×10个拷贝。以其他六种猪病毒作为模板测试了该检测方法的特异性，未显示任何交叉反应。使用所开发的mRT-PCR检测方法对总共280份现场样本进行了检测。发现SaV、EMCV、RVA和AstV的阳性率分别为24.6%（69/280）、5%（14/280）、4.3%（12/280）和17.5%（49/280）。与对每种病毒进行单独检测相比，这种mRT-PCR检测方法是一种用于检测SaV、EMCV、RVA和AstV混合感染或单一感染的简单、快速且经济高效的方法。
