数字PCR检测HIV-1完整前病毒DNA方法的建立  

Establishment of droplet digital PCR for detected HIV-1 intact proviral DNA

作　　者：何林[1] 劳晓洁 张媛媛 梁淑家[4] 李丹[1] 廖玲洁[1] 邢辉[1] 洪坤学[1] 马丽英[1] He Lin;Lao Xiaojie;Zhang Yuanyuan;Liang Shujia;Li Dan;Liao Lingjie;Xing Hui;Hong Kunxue;Ma Liying;无(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Beijing Key Laboratory of Emerging Infectious Diseases,Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China;Beijing Institute of Infectious Diseases,Beijing 100015,China;Guangxi Key Laboratory of Major Infectious Disease Prevention Control and Biosafety Emergency Response,Guangxi Center for Disease Control and Prevention,Nanning 530028,China)

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,传染病溯源预警与智能决策全国重点实验室,北京102206 [2]首都医科大学附属北京地坛医院传染病研究所,新发突发传染病研究北京市重点实验室,北京100015 [3]北京市感染性疾病研究中心,北京100015 [4]广西壮族自治区疾病预防控制中心,广西重大传染病防控与生物安全应急响应重点实验室,南宁530028

出　　处：《中华实验和临床病毒学杂志》2024年第1期86-92,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金(81871694,81561128006)。

摘　　要：目的通过数字PCR建立检测HIV-1完整前病毒DNA方法。方法通过培养8E5细胞(含有单拷贝整合HIV-1前病毒的T淋巴细胞系),提取DNA。通过连续稀释DNA,利用数字PCR扩增1倍、5倍、25倍、625倍、3125倍和15625倍稀释的HIV-1Ψ区、env区和真核细胞10号染色体RPP30区,计算线性关系及最低检测浓度。在数字PCR体系中分别加入5μl、3.1μl、2.5μl的DNA,各进行2批次检测,每批次重复检测4次,判断其批内变异系数,同核酸量和不同核酸量的批间变异系数判断稳定性。利用8E5 DNA检测细胞中完整前病毒含量。结果DNA在6个稀释度下,Ψ区、env区和RPP30区线性拟合优度分别是R^(2)≥0.999、R^(2)≥0.993、R^(2)≥0.996。当稀释到3125倍时,Ψ区、env区和RPP30区最低阳性液滴检出3个、2个和2个,检出浓度是2.37拷贝/μl、1.21拷贝/μl和1.58拷贝/μl。DNA的数字PCR重复性实验检测,Ψ区批内变异系数从0.66%到3.43%,同核酸量的批间变异系数分别是3.19%、4.3%和3.45%,不同核酸量的批间变异系数仅4.35%。env区批内变异系数从0.7%到3.2%,同核酸量的批间变异系数分别是3.18%、4.52%和3.4%,不同核酸量的批间变异系数仅4.02%。RPP30区批内变异系数从0.91%到2.91%,同核酸量的批间变异系数分别是3%、4.55%和3.37%,不同核酸量的批间变异系数仅在3.98%。8E5细胞中含缺陷前病毒的比例和含完整前病毒的比例分别是90%和45%。结论利用数字PCR能够检测HIV-1完整前病毒DNA,表现出较强的稳定性,为HIV-1感染者储存库检测提供技术手段。Objective To establishment an assay for HIV-1 intact proviral DNA assay through droplet digital PCR(ddPCR).Methods DNA was extracted by culturing 8E5 cells,a Tlymphocyte cell line containing a single copy of integrated HIV-1 provirus.Serial diluting DNA were prepared by amplified 1-fold,5-fold,25-fold,625-fold,3125-fold,and 15625-fold across the HIV-1Ψregion,env region,and eukaryotic chromosome 10 RPP30 regions,and the linear relationship was calculated and the minimum detection concentration.DNA solution of 5μl,3.1μl,2.5μl was added to the ddPCR mixture respectively,with each dilution undergoing two batches of detection,and each was repeated four times.The intra-batch variation coefficient was detected,while the inter-batch variation coefficient was detected by the same DNA amount and different DNA amounts to determine the stability;8E5 cell was used to detect the intact proviral content in cells.Results The linear fitting goodness ofΨregion,env region and RPP30 region are R^(2)≥0.999,R^(2)≥0.993,R^(2)≥0.996 in 6 dilutions of DNA,respectively.At a 3125-fold dilution,the lowest positive droplets were detected in theΨregion,env region and RPP30 region were 3,2 and 2,respectively,the detected concentrations were 2.37 copies/μl,1.21 copies/μl and 1.58 copies/μl.The ddPCR repeatability experimental detecting DNA showed that theΨregion of the intra-batch variation coefficients ranged from 0.66%to 3.43%,with the inter-batch variation coefficients of the same DNA at 3.19%,4.3%and 3.45%respectively,and the inter-batch variation coefficients of the different DNA at only 4.35%.The env region of the intra-batch variation coefficients ranged from 0.7%to 3.20%,with the inter-batch variation coefficients of the same DNA at 3.18%,4.52%and 3.4%respectively,and the inter-batch variation coefficients of the different DNA at only 4.02%.The RPP30 region of the intra-batch variation coefficients ranged from 0.91%to 2.91%,with the inter-batch variation coefficients of the same DNA at 3%,4.55%and 3.37%respectively,and t

关 键 词：HIV-1 数字PCR IPDA 稳定性 线性性能 

分 类 号：R440[医药卫生—诊断学] R512.91[医药卫生—临床医学]