猪内源性逆转录病毒检测的三重PCR方法的建立及其在五指山猪组织样品检测中的初步应用

doi:10.11843/j.issn.0366-6964.2025.07.046

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (7): 3548-3554.doi: 10.11843/j.issn.0366-6964.2025.07.046

猪内源性逆转录病毒检测的三重PCR方法的建立及其在五指山猪组织样品检测中的初步应用

瑞雪¹^,²^,³(), 张云航²^,³, 李杨²^,³, 谭晨²^,³, 蔡艺菲²^,³, 刘园园¹^,²^,³, 曹宗喜¹^,², 张艳¹^,², 孙瑞萍¹^,², 刘光亮¹^,²^,³^,*()

1. 海南省农业科学院畜牧兽医研究所海南省热带动物繁育与疫病研究重点实验室，海口 571100
2. 新疆农业大学动物医学学院，乌鲁木齐 830000
3. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室，兰州 730030

收稿日期:2024-08-21 出版日期:2025-07-23 发布日期:2025-07-25
通讯作者: 刘光亮 E-mail:905500891@qq.com;LiuGuangliang01@caas.cn
作者简介:瑞雪(1996-)，女，内蒙古兴安盟人，硕士生，主要从事动物疫苗与分子免疫学研究, E-mail: 905500891@qq.com
基金资助:
科技部重点研发计划前沿生物技术专项课题(2021YFA0805905)；海南省重大科技计划项目(ZDKJ2021030)；国家重点研发计划课题(2023YFC3404302)

Establishment of a Triple PCR Method for the Detection of Porcine Endogenous Retroviruses and Its Preliminary Application to the Detection of Wuzhishan Pig Tissue Samples

RUI Xue¹^,²^,³(), ZHANG Yunhang²^,³, LI Yang²^,³, TAN Chen²^,³, CAI Yifei²^,³, LIU Yuanyuan¹^,²^,³, CAO Zongxi¹^,², ZHANG Yan¹^,², SUN Ruiping¹^,², LIU Guangliang¹^,²^,³^,*()

1. Institute of Animal Husbandry and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Hainan Provincial Key Laboratory of Tropical Animal Breeding and Disease Research, Haikou 571100, China
2. Xinjiang Agricultural University, Urumqi 830000, China
3. State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730030, China

Received:2024-08-21 Online:2025-07-23 Published:2025-07-25
Contact: LIU Guangliang E-mail:905500891@qq.com;LiuGuangliang01@caas.cn

摘要/Abstract

摘要：

本研究旨在建立可同时检测猪基因组中的猪内源性逆转录病毒(porcine endogenous retrovirus，PERV)并鉴别PERV-A、PERV-B、PERV-C三种亚型PERV的三重PCR检测方法。根据PERV不同亚型囊膜(env)基因设计特异性引物，经反应条件和反应体系优化后对该方法进行特异性、敏感性及重复性试验验证。结果表明：本研究建立的三重PCR检测方法特异性强，可特异性扩增三种亚型PERV目的基因，对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒扩增结果呈阴性；该方法的敏感性较好，对PERV-A、PERV-B、PERV-C质粒标准品的检测下限分别为1×10²、1×10³和1×10²拷贝·μL^-1；采用建立的三重PCR方法检测103份组织样品，其中PERV-A的阳性率为100%(103/103)，PERV-B的阳性率为100%(103/103)，PERV-C的阳性率为54%(56/103)，与经典单重PCR方法进行比较，符合率分别为100%、100%、96.6%。本研究成功建立了一种特异性强、敏感性较好、快速简便的三重PCR方法，可用于PERV三种亚型同时检测的鉴别诊断，为筛选无PERV的猪源供体器官提供技术支撑。

关键词: 猪内源性逆转录病毒, 囊膜基因, 三重PCR检测方法, 组织样品

Abstract:

This study aims to establish a preliminary triplex PCR assay capable of simultaneously detecting porcine endogenous retroviruses (PERV) in the pig genome and differentiating the three PERV subtypes: PERV-A, PERV-B, and PERV-C. Specific primers were designed based on the envelope (env) gene of different PERV subtypes. The method was validated for specificity, sensitivity and reproducibility after optimization of the reaction conditions and the reaction system. The results showed that the triplex PCR method specifically amplified all three PERV subtypes. The detection limits for the plasmid standards of PERV-A, PERV-B, and PERV-C were 1.50×10², 1.99×10³, and 1.57×10² copies·μL^-1, respectively. A total of 103 tissue samples were tested using the triplex PCR method and compared with the conventional single PCR method. The positive rates were 100% (103/103) for PERV-A, 100% (103/103) for PERV-B and 54% (56/103) for PERV-C. The compliance rates for the two methods were PERV-A, PERV-B, and PERV-C were 100%, 100%, and 96.6%, respectively. This study successfully developed a highly specific, sensitive, rapid, and convenient triplex PCR assay, which can be used for the simultaneous detection and differential diagnosis of the three PERV subtypes, and provides technical support for the screening of PERV-free porcine-derived donor organs.

Key words: porcine endogenous retrovirus, env gene, triple PCR method, tissue samples

中图分类号:

S854.43

瑞雪, 张云航, 李杨, 谭晨, 蔡艺菲, 刘园园, 曹宗喜, 张艳, 孙瑞萍, 刘光亮. 猪内源性逆转录病毒检测的三重PCR方法的建立及其在五指山猪组织样品检测中的初步应用[J]. 畜牧兽医学报, 2025, 56(7): 3548-3554.

RUI Xue, ZHANG Yunhang, LI Yang, TAN Chen, CAI Yifei, LIU Yuanyuan, CAO Zongxi, ZHANG Yan, SUN Ruiping, LIU Guangliang. Establishment of a Triple PCR Method for the Detection of Porcine Endogenous Retroviruses and Its Preliminary Application to the Detection of Wuzhishan Pig Tissue Samples[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3548-3554.

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名称 Name	引物序列(5′→3′) Primer sequence	片段大小/bp Produce size
共用上游引物Share upstream primer	CATCGCTTGGTTCCTTACTCTGTC
PERV-AR	CTTCTCCCAGTGCCTCCATAGT	629
PERV-BR	TGTTGCTAGGCGGCTGTGTTATGT	743
PERV-CR	TTCCAGTTCTAATCCAGGTCAGGT	462

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猪内源性逆转录病毒检测的三重PCR方法的建立及其在五指山猪组织样品检测中的初步应用

Establishment of a Triple PCR Method for the Detection of Porcine Endogenous Retroviruses and Its Preliminary Application to the Detection of Wuzhishan Pig Tissue Samples

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病毒名称 Virus name	多重PCR检测方法 Multi PCR detection method		单重PCR检测方法 Single PCR detection method		阳性符合率/% Positive accordance rate
病毒名称 Virus name	阳性/样本量 Positive samples/Total samples	阳性率/% Positive rate	阳性/样本量 Positive samples/Total samples	阳性率/% Positive rate	阳性符合率/% Positive accordance rate
PERV-A	103/103	100	103/103	100	100
PERV-B	103/103	100	103/103	100	100
PERV-C	56/103	54	58/103	56	96.6