整合型HIV DNA荧光定量检测法的临床初探  

Application of the fluorescence quantitative method to detect the integrated HIV DNA in the clinical study

作　　者：张敏[1] 王前英 李昕[1] 胡芸文[1] 施碧胜[2] 

机构地区：[1]上海市公共卫生临床中心医学检验科,201508 [2]上海市公共卫生临床中心科学研究部,201508

出　　处：《中华实验和临床病毒学杂志》2017年第6期566-569,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：上海市卫生和计划生育委员会科研课题（20144Y0075）

摘　　要：目的 建立外周血单核细胞整合型HIV DNA荧光定量ALu-PCR的检测方法.方法 收集我院就诊的病毒载量＜ 20IU/ml的艾滋病患者、耐药患者和初诊患者各10例.培养ACH2细胞系,每个细胞含有一个HIV基因组.构建pMD19T-CD3质粒,测定浓度,计算其拷贝数.选用针对HIV3＇长末端重复序列的引物ULF1,和针对人类基因高度保守的Alu1和Alu2序列的引物,对1∶5梯度稀释,共8个梯度的ACH2基因组及样本进行预扩增.将预扩增的产物用引物Lambda T、UR2及HIV Taqman探针进行整合型HIV DNA的荧光扩增;用引物CD3IN5、CD3IN3及CD3 Taqman探针对预扩增产物及1∶5梯度稀释的CD3质粒进行CD3荧光扩增.结果 以1∶5梯度稀释的CD3质粒为标准品,得到的线性回归方程y=-2.731x ＋43.01,（R2 =0.953）,根据一个人类细胞含2个CD3基因,可得到样本及ACH2的细胞数.ACH2的细胞数即HIV拷贝数为4.271×104;以1∶5梯度稀释的ACH2为标准品,回归方程为y=-3.146x ＋39.11 （R2 =0.968）计算可得样本整合型HIV DNA;用样本的整合型HIV-1 DNA/样本的CD3的拷贝数,即为单位细胞中整合型HIV拷贝数.通过对不同组间的t检验,各组间无差异（P＞0.05）.结论 该方法对于低拷贝HIV病毒量的检测有优势,提示通常的抗病毒治疗无法有效攻击潜伏库中的病毒,同时也为新的干预治疗提供了一种评价依据.Objective To establish fluorescence quantitative polymerase chain reaction （PCR） to detect integrated HIV DNA in peripheral blood mononuclear cells.Methods A total of 30 HIV-seropositve individuals were enrolled in this study,including 10 subjects with a detection limit of 20 copies/ml of plasma,10 patients with drug resistance and 10 patients with no history of antiretroviral therapy （ART）.Cultivated ACH2 cells carried a single copy of the integrated HIV genome.We have built pMD19T-CD3 plasmid and calculated the copy number.We used oligonucleotides ULF1 specific for the long terminal repeats （LTR） regions and two oliligonucleotides specific for human Alu sequences to pre-amplified the integrated HIV DNA.Samples and serial dilutions of ACH2 cells were all pre-amplified,the products of which were used for the second round fluorescence amplifications.The Lambda T primers,UR2 primers and HIV Taqman probes were used for second round amplifications in integrated HIV DNA assay.The CD3IN5 primers,CD3IN3 primers and CD3 Taqman probes were used for CD3 quantification.Results Serial 5-fold dilutions of the plasmid were used as standards for CD3 gene quantifications.The equation of the linear regression was y =-2.731x ＋43.01 （R2 =0.953）.The cellular input was quantified by number of human genome equivalents （CD3 gene,2 copies/cell）.The copies of ACH2 cells was 4.271 × 104,which was the cellular input of the ACH2 cells.Serial 5-fold dilutions of ACH2 cells were used to generate a standard curve for the integrated HIV DNA assays.The equation of the linear regression was y =-3.146x ＋ 39.11 （R2 =0.968）.The ratio between the number of copies of the integrated HIV DNA form and the number of cells （CD3 copies） was calculated to obtain the frequency of cells.Based on the test between different groups,each group had no difference （P ＞ 0.05）.Conclusions This method presented advantage in detection of the lower copies of virus.It hinted that the current antiretroviral therapy can not effectively

关 键 词：HIV 荧光定量PCR ACH2细胞 CD3基因 

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