文献类型： 外文期刊

第一作者： Xin, Zhonghao

作者： Xin, Zhonghao;Li, Shiheng;Lu, Xiao;Liu, Liping;Gao, Yuehua;Hu, Feng;Yu, Kexiang;Ma, Xiuli;Li, Yufeng;Huang, Bing;Guo, Xiaozhen;Li, Shiheng;Lu, Xiao;Wu, Jiaqiang

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关键词： TaqMan probe; multiplex real-time RT-PCR; porcine enteroviruses; diagnosis

期刊名称：VETERINARY SCIENCES （ 影响因子：2.0； 五年影响因子：2.2 ）

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年卷期： 2024 年 11 卷 7 期

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收录情况： SCI

摘要： Simple Summary Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four significant pathogens of viral diarrhea of piglets in large-scale pig farms, which have caused huge economic losses to the pig industry all over the world. Since these four viruses have very similar clinical symptoms, it is necessary to develop an excellent detection method that can differentiate and diagnose these four viruses. In this study, we developed a multiplex real-time RT-PCR method that can simultaneously differentiate and diagnose these four viruses. The method has high specificity and does not cross-react with other porcine viruses. The lower limit of detection was 2.18 x 102 copies/mu L. In addition, we tested 97 clinical samples collected by this method, and the results were consistent with the detection results of traditional RT-PCR, indicating that this method is reliable. In summary, we 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 diarrhea virus diseases.Abstract 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 x 102 copies/mu L 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.

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