He Junxian, Shi Kaichuang, Shi Yuwen, Yin Yanwen, Feng Shuping, Long Feng, Qu Sujie, Song Xingju
College of Animal Science and Technology, Guangxi University, Nanning 530005, China.
Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
Pathogens. 2024 Nov 29;13(12):1052. doi: 10.3390/pathogens13121052.
Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09-1.24% for intra-assay and 0.08-1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases.
猪星状病毒（PoAstV）、猪萨波病毒（PoSaV）、猪诺如病毒（PoNoV）和猪轮状病毒A（PoRVA）是新发现的重要猪腹泻病毒，宿主范围广泛且具有人畜共患病潜力，在猪群中常发现它们的共同感染。本研究针对PoAstV、PoSaV和PoNoV的ORF1基因以及PoRVA的VP6基因设计了特异性引物和探针。构建了重组标准质粒，优化了反应条件（引物和探针浓度、退火温度和反应循环），并分析了特异性、敏感性和重复性，以建立一种用于检测这四种腹泻病毒的四重实时定量RT-PCR（RT-qPCR）检测方法。结果表明，该检测方法能有效检测PoAstV、PoSaV、PoNoV和PoRVA，与其他猪病毒无交叉反应，对PoAstV、PoSaV、PoNoV和PoRVA的检测限（LOD）分别为138.001、135.167、140.732和132.199（拷贝/反应），具有高特异性和敏感性。此外，它还具有良好的重复性，批内变异系数（CV）为0.09 - 1.24%，批间变异系数为0.08 - 1.03%。采用所建立的检测方法对来自中国广西14个城市的1578份临床粪便样本进行了分析。结果表明，广西的临床样本中PoAstV的流行率为35.93%（567/1578），PoSaV为8.37%（132/1578），PoNoV为2.98%（47/1578），PoRVA为14.32%（226/1578），混合感染的发生率显著，为18.31%（289/1578）。同时，用先前建立的检测方法对这1578份临床样本进行了分析，两种方法的符合率超过99.43%。本研究开发了一种高效、精确的诊断方法，用于检测和区分PoAstV、PoSaV、PoNoV和PoRVA，能够成功诊断这四种疾病。
