Xin Zhonghao, Li Shiheng, Lu Xiao, Liu Liping, Gao Yuehua, Hu Feng, Yu Kexiang, Ma Xiuli, Li Yufeng, Huang Bing, Wu Jiaqiang, Guo Xiaozhen
Key Laboratory of Poultry Disease Diagnosis and Immunity in Shandong Province, Poultry Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China.
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010020, China.
Vet Sci. 2024 Jul 9;11(7):305. doi: 10.3390/vetsci11070305.
Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 10 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.
猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、猪德尔塔冠状病毒（PDCoV）和猪轮状病毒A（PoRVA）是引起猪病毒性腹泻的四种主要病原体，它们常混合感染，仅根据临床症状难以区分。在此，我们首次开发了一种基于多重TaqMan探针的实时RT-PCR方法，用于同时检测PEDV、TGEV、PDCoV和PoRVA。针对PEDV的M蛋白基因、TGEV的N蛋白基因、PDCoV的N蛋白基因和PoRVA的VP7蛋白基因设计了特异性引物和探针，并构建了相应的重组质粒。该方法具有极高的特异性、高灵敏度和出色的重复性；在多重实时RT-PCR检测中，检测限（LOD）可低至2.18×10拷贝/μL。共使用97份临床样本比较传统逆转录PCR（RT-PCR）和这种多重实时RT-PCR检测PEDV、TGEV、PDCoV和PoRVA的结果，结果完全一致。随后，将随机选取的5份检测呈阳性的临床样本送去进行DNA测序验证，测序结果与传统RT-PCR及本研究开发方法的检测结果一致。综上所述，本研究开发了一种同时检测PEDV、TGEV、PDCoV和PoRVA的多重实时RT-PCR方法，本研究结果可为这四种猪病毒性腹泻疾病的鉴别诊断和流行病学调查提供技术手段。
