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首页 > 过刊浏览>2021年第31卷第9期 >90-97. DOI:10. 3969 / j.issn.1671-7856. 2021. 09. 014
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基于微滴式数字 PCR 技术对猪内源逆转录病毒拷贝数的检测方法的建立及应用
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中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,国家卫生健康委员会人类疾病比较医学重点实验室, 国家中医药管理局人类疾病动物模型三级实验室,北京 100021

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R-33


Establishment and application of a droplet digital PCR-based method for measuring PERV copy number in the porcine genome
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  • LU Tianyu

    LU Tianyu

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • GAO Hong

    GAO Hong

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • YANG Bochao

    YANG Bochao

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • YU Jianan

    YU Jianan

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • XU Yaxin

    XU Yaxin

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • WANG Ruolin

    WANG Ruolin

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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  • QIN Chuan

    QIN Chuan

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China
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Affiliation:

Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Comparative Medicine Center, Peking Union Medical College (PUMC), Key Laboratory of Human Disease Comparative Medicine, National Health Commission of the People’s Republic of China; Key Laboratory of Human Disease Animal Models, State Administration of Traditional Chinese Medicine, Beijing 100021, China

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    摘要:

    目的 利用微滴式数字 PCR( droplet digital PCR, ddPCR)技术,对猪基因组中猪内源逆转录病毒 (porcine endogenous retrovirus, PERV) 拷贝数测定方法进行优化,建立 PERV 拷贝数的检测方法。 方法 采用 TaqMan 探针双重 ddPCR方法,分别以甘油醛-3-磷酸脱氢酶( glyceraldehyde-3-phosphate dehydrogenase, GAPDH)和转铁蛋白受体(transferrin receptor, TFRC)为内参基因,确定对 PERV 拷贝数检测反应最适合退火温度和反应循环数。另外,通过对浓度梯度稀释的标准品质粒和猪细胞基因组进行检测,以确定最适模板浓度。采用 ddPCR 和 qPCR 两种方法测定来自猪源细胞系和巴马小型猪组织中 PERV 的拷贝数,比较两种检测方法的稳定性。 结果 基于 ddPCR 技术检测 PERV 拷贝数的最适退火温度为 59. 9℃ ,最佳反应循环数为 50;模板基因组的最适浓度在 1. 25~ 7. 5 ng;检测同一样品来源基因组中 PERV 的拷贝数的变异系数在 0. 44% ~ 8. 29%。 结论 本研究建立了基于 ddPCR 技术的 PERV 拷贝数的检测方法并系统的探索了最佳实验条件,与传统的 qPCR 相比,该方法具有更高的准确性和可重复性,为异种移植研究中供体动物基因组 PERV 拷贝数的测定提供了灵敏的方法和可靠依据。

    Abstract:

    Objective To optimize the experimental conditions for the determination of porcine endogenous retrovirus (PERV) copy number in the porcine genome using a droplet digital polymerase chain reaction ( ddPCR) technique. Methods TaqMan probe duplex-ddPCR was used to determine the copy number of PERV, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) and transferrin receptor protein 1 ( TFRC) were adopted as the internal reference genes. To determine the optimal annealing temperature and PCR cycle number, we compared multiple conditions. In addition, the suitable range of template amount was estimated by examining the serially diluted plasmid and the pig cell genome. Genomic DNA isolated from pig cell lines or Bama minipig tissue was used to determine the copy number of PERV using ddPCR-and quantitative PCR-based method, which demonstrated the repeatability of different method. Results The optimal annealing temperature for the ddPCR-based method to measure PERV copy number was 59. 9℃ , and the optimal number of PCR cycles was 50. The suitable template genome concentration ranged from 1. 25 to 7. 5 ng. The variation of coefficients for PERV copy number in the porcine genome ranged from 0. 44% to 8. 29%. Conclusions A method for analyzing the PERV copy number based on the ddPCR technique was established, which improved the accuracy and repeatability compared with a traditional quantitative PCR-based detection approach. This method provides a sensitive approach and is a reliable basis for the determination of PERV copy number in donor animal genomes for xenotransplantation studies.

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卢天宇,高 虹,杨博超,于佳楠,徐亚新,王若琳,秦 川.基于微滴式数字 PCR 技术对猪内源逆转录病毒拷贝数的检测方法的建立及应用[J].中国比较医学杂志,2021,31(9):90~97.

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  • 收稿日期:2020-12-01
  • 在线发布日期: 2021-10-25
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