艾滋病模型中关键指标SIV DNA绝对定量微滴式数字PCR技术的创新应用  

The establishment and application of an absolute quantitative method for analyzing SIV DNA via droplet digital PCR

作　　者：张丽丽 陆佳涵 薛婧 丛喆 魏强 ZHANG Lili;LU Jiahan;XUE Jing;CONG Zhe;WEI Qiang(Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences(CAMS),Comparative Medicine Center,Peking Union Medical College(PUMC),NHC Key Laboratory of Human Disease Comparative Medicine,Key Laboratory of Human Diseases Animal Models,State Administration of Traditional Chinese Medicine,Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases,Beijing 100021,China)

机构地区：[1]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,国家卫健委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,新发再发传染病动物模型研究北京市重点实验室,北京100021

出　　处：《中国比较医学杂志》2020年第5期27-30,53,共5页Chinese Journal of Comparative Medicine

基　　金：中国医学科学院医学与健康科技创新工程重大协同创新项目(2017-I2M-1-014);十三五传染病科技重大专项(2017ZX10304402,2018ZX10101001,2018ZX10301103);国家自然科学基金(81971944)。

摘　　要：目的建立微滴式数字PCR技术(droplet digital PCR,ddPCR)作为关键指标,实现精准检测艾滋病动物模型中细胞及组织中SIV DNA载量,用于储存库隐匿病毒实时动态评估。方法根据成熟的qPCR方法,对微滴式dPCR的反应条件进行了优化,确定最适退火温度;通过对10倍系列稀释pGEM-SIVgag477质粒标准品的检测,确定ddPCR检测范围。并通过对若干DNA样品的多次检测,判断该检测方法的稳定性。结果ddPCR的退火温度为60℃;检测范围是0.3~3.96×104 copies/μL;微滴式dPCR与qPCR有良好的相关性,相关系数r=0.9981。结论本研究建立了基于微滴式dPCR的SIV病毒DNA的绝对定量方法,可有效的对细胞和组织中病毒储存库样本中病毒DNA载量进行动态绝对定量,极大提高了模型应用评价的准确度。Objective To establish a sensitive droplet digital PCR(ddPCR)assay for measuring the proviral DNA load in PBMCs(peripheral blood mononuclear cell)and tissues from SIV-infected animals.Methods According to the standard qPCR method,we first optimized the annealing temperature of the droplet dPCR,and then determined the detection range of the ddPCR.This was followed by correlation analysis of ddPCR and qPCR via detection of the 10-fold serially diluted pGEM-SIVgag477.We repeatedly tested 7 DNA samples extracted from PBMCs of SIV-infected monkeys to study the repeatability of the method.Results The annealing temperature of the ddPCR was determined to be 60℃ and the detection range was 0.3~3.96×104 copies/μL.The correlation coefficient of ddPCR and qPCR was in the range of 0.9981.Conclusions We established an absolute quantitative method for SIV DNA analysis based on droplet dPCR.This method can be used to accurately quantify the proviral DNA load and represents an effective technique for the measurement of viral reservoirs in SIV research.

关 键 词：微滴式数字PCR 猴免疫缺陷病毒 DNA 绝对定量 

分 类 号：R-33[医药卫生]