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

Acta Veterinaria et Zootechnica Sinica ›› 2025, Vol. 56 ›› Issue (7): 3548-3554.doi: 10.11843/j.issn.0366-6964.2025.07.046

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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

Abstract

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

CLC Number:

S854.43

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.

Figures/Tables 6

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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

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