PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2024.05.044

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2267-2272.doi: 10.11843/j.issn.0366-6964.2024.05.044

PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用

胡泽奇¹, 李润成¹, 谭祖明³, 谢秀艳¹, 王江平², 秦乐娟², 李荣^2*, 葛猛^1*

1. 湖南农业大学动物医学院, 长沙 410000;
2. 湖南龙华农牧发展有限公司, 茶陵 412400;
3. 茶陵县畜牧水产事务中心, 茶陵 412400

收稿日期:2023-07-20 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 李荣，主要从事规模化猪场管理与疫病防控，E-mail：120547112@qq.com;葛猛，主要从事动物疫病诊断及防控研究，E-mail：gmg02@126.com
作者简介:胡泽奇(2000-)，男，湖南涟源人，硕士生，主要从事动物疫病诊断及防控研究，E-mail：2878815126@qq.com
基金资助:
湖南省重点研发计划项目(2023NK2017);云南省重大科技专项计划(202202AE090032); 湖南省教育厅重点项目(21A0125)；湖南省普通高等学校科技创新团队支持计划资助

Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay

HU Zeqi¹, LI Runcheng¹, TAN Zuming³, XIE Xiuyan¹, WANG Jiangping², QIN Lejuan², LI Rong^2*, GE Meng^1*

1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410000, China;
2. Hunan Longhua Agriculture and Animal Husbandry Development Co., LTD, Chaling 412400, China;
3. Chaling County Livestock and Aquatic Affairs Center, Chaling 412400, China

Received:2023-07-20 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 旨在建立一种可快速同时检测猪流行性腹泻病毒 (porcine epidemic diarrhea virus, PEDV)、猪A群轮状病毒 (porcine rotavirus type A, PoRVA) 和猪丁型冠状病毒 (porcine deltacoronavirus, PDCoV) 三重荧光定量PCR方法。针对PEDV-N、PoRVA-VP6和PDCoV-M基因设计了引物和探针，条件优化后进行性能评估，并与商品化试剂盒检测对比。结果显示：本研究建立的三重RT-qPCR方法具有良好特异性，对PRRSV、PCV₂和PRV等阳性核酸不发生扩增；具有较高的敏感性，PEDV、PoRVA和PDCoV的最低检测限均达1 copies·μL^-1；重复性良好，组内和组间变异系数均小于1%；样本适应性广，检测不同样本类型时，变异系数均小于1%。与商品化试剂盒对比，本研究建立的方法PEDV和PoRVA的检测符合率为92.5%和97.5%，并对PDCoV的检测范围更广，对其变异毒株仍有较好的检测效果。本研究建立的检测方法具有特异性好、敏感性高、稳定性强和样本适应性广等优势，为临床腹泻样本提供了一种好的检测方法。

关键词: 猪流行性腹泻病毒, 猪A群轮状病毒, 猪丁型冠状病毒, 三重RT-qPCR

Abstract: This paper aims to establish a triple fluorescence quantitative PCR method for the simultaneous detection of porcine epidemic diarrhea virus (PEDV), porcine rotavirus type A (PoRVA) and porcine deltacoronavirus (PDCoV). Primers and probes were designed for PEDV-N gene, PoRVA-VP6 gene and PDCoV-M gene, and their performance was evaluated after the conditions were optimized, and compared with commercial kits. The results showed that the triple RT-qPCR method established in this study had good specificity and did not amplify positive nucleic acids such as PRRSV, PCV2 and PRV. The detection limits of PEDV, PoRVA and PDCoV were all 1 copies·μL^-1. The coefficient of variation within and between groups was less than 1%. The sample has wide adaptability, and the coefficient of variation is less than 1% when different sample types are detected. Compared with commercial kits, the detection coincidence rates of PEDV and PoRVA were 92.5% and 97.5%, and the detection range of PDCoV was wider, and the detection effect of PDCoV variant strains was still good. The detection method established in this study has the advantages of good specificity, high sensitivity, strong stability and wide sample adaptability, which provides a good detection method for clinical diarrhea samples.

Key words: porcine epidemic diarrhea virus, porcine rotavirus type A, porcine deltacoronavirus, triple RT-qPCR

中图分类号:

S858.285.3

胡泽奇, 李润成, 谭祖明, 谢秀艳, 王江平, 秦乐娟, 李荣, 葛猛. PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 2024, 55(5): 2267-2272.

HU Zeqi, LI Runcheng, TAN Zuming, XIE Xiuyan, WANG Jiangping, QIN Lejuan, LI Rong, GE Meng. Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2267-2272.

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PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用

Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay

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