摘要
目的利用微滴式数字PCR(droplet digital PCR,dd PCR)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Taq Man探针双重dd PCR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dd PCR和q PCR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dd PCR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dd PCR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q PCR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。
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( dd PCR)technique. Methods Taq Man 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 dd PCR-and quantitative PCR-based method,which demonstrated the repeatability of different method. Results The optimal annealing temperature for the dd PCR-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 dd PCR 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.
作者
卢天宇
高虹
杨博超
于佳楠
徐亚新
王若琳
秦川
LU Tianyu;GAO Hong;YANG Bochao;YU Jianan;XU Yaxin;WANG Ruolin;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)
出处
《中国比较医学杂志》
CAS
北大核心
2021年第9期90-97,共8页
Chinese Journal of Comparative Medicine
基金
中国医学科学院医学创新工程重大协同创新项目(2017-I2M-2-005)。
关键词
微滴式数字PCR
猪内源逆转录病毒
拷贝数
绝对定量
droplet digital PCR
porcine endogenous retrovirus
copy number
absolute quantification
