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、3 125倍和15 625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5 μl、3.1 μl、2.5 μl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R2≥0.999、R2≥0.993、R2≥0.996.当稀释到3 125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.
Abstract
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,3 125-fold,and 15 625-fold across the HIV-1 Ψ region,env region,and eukaryotic chromosome 10 RPP30 regions,and the linear relationship was calculated and the minimum detection concentration.DNA solution of 5 μl,3.1 μl,2.5 μl 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 of Ψ region,env region and RPP30 region are R2 ≥0.999,R2 ≥ 0.993,R2 ≥ 0.996 in 6 dilutions of DNA,respectively.At a 3 125-fold dilution,the lowest positive droplets were detected in the Ψ region,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 the Ψ region 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 the inter-batch variation coefficients of the different DNA at only 3.98％.The proportion of 8E5 cells containing defective provirus and the proportion of intact provirus were calculated to be approximately 90％and 45％,respectively.Conclusions Droplet digital PCR used to detect HIV-1 intact proviral DNA,showed strong stability and provided a technical means for HIV-1 infection reservoir detection.
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