Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

中文翻译：

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区分 TGEV、PEDV、PDCoV 和 PoRVA 的四重实时 RT-qPCR 检测方法的建立和应用

猪病毒性腹泻是临床上的常见疾病，给养猪业造成重大经济损失。猪病毒性腹泻的罪魁祸首包括传染性胃肠炎病毒（TGEV）、猪流行性腹泻病毒（PEDV）、猪三角洲冠状病毒（PDCoV）和猪轮状病毒A（PoRVA）。涉及病毒的合并感染在临床环境中很常见，从而增加了鉴别诊断的复杂性。因此，有必要开发一种能够以高灵敏度和特异性检测和区分所有四种猪腹泻病毒（TGEV、PEDV、PDCoV 和 PoRVA）的方法。目前，聚合酶链反应（PCR）是病原体检测的首选方法。与传统 PCR 相比，TaqMan 实时 PCR 具有更高的灵敏度、卓越的特异性和准确性。本研究旨在开发一种四重实时 RT-qPCR 检测方法，利用 TaqMan 探针，对 TGEV、PEDV、PDCoV 和 PoRVA 进行独特检测。本研究中设计的四重实时 RT-qPCR 检测显示出能够避免检测到不相关病原体的能力，并表现出值得称赞的特异性、灵敏度、重复性和再现性，检测限 (LOD) 为 27 拷贝/微升。在涉及 5483 个临床样本的对比分析中，商业 RT-qPCR 试剂盒和四重 RT-qPCR 对 TGEV、PEDV、PDCoV 和 PoRVA 检测的结果完全一致。在广西壮族自治区 10 月至 3 月采集样本后，我们评估了仔猪腹泻样本中 TGEV、PEDV、PDCoV 和 PoRVA 的流行情况，结果显示阳性检出率为 0.2% (11/5483)、8.82% (485/5483)。 ）、1.22% (67/5483) 和 4.94% (271/5483)。 PEDV/PoRVA、PEDV/PDCoV、TGEV/PED/PoRVA 和 PDCoV/PoRVA 的合并感染率分别为 0.39%、0.11%、0.01% 和 0.03%，未检测到其他合并感染，如下所示：通过四重实时 RT-qPCR 测定。这项研究不仅为在实际应用中同时区分 TGEV、PEDV、PDCoV 和 PoRVA 建立了一个有价值的工具，而且还为了解广西这些引起腹泻的病毒病原体的流行情况提供了重要的见解。