磁珠富集法提高外周血HIV DNA检测灵敏度的研究

Enhancing the sensitivity of HIV DNA detection:a magnetic bead-based method

目的建立外周血人免疫缺陷病毒脱氧核糖核酸(HIV DNA)磁珠富集检测方法,并评估该方法在提高HIV感染患者早期抗病毒治疗(early antiretrovital treatment,ART)后外周血中HIV DNA检测灵敏度中的应用价值。方法采集1位HIV感染窗口期(window period,WP)即开展ART患者4个不同时间点的外周全血,Ficoll法分离外周全血单个核细胞(peripheral blood mononuclear cells,PBMC),阴性分选富集PBMC中CD4^(+)T淋巴细胞,应用HIV DNA检测试剂盒检测磁珠富集组和全血组HIV DNA浓度。结果采用免疫磁珠富集法从PBMC中分离纯化CD4^(+)T细胞,经流式细胞仪测定纯度达到(96.4±2.6)%,0.4%台盼兰染色细胞活力为(95.9±2.9)%。治疗后,在追踪的不同时间点,发现患者血浆中HIV RNA载量逐渐下降,至第4个追踪时间点,血浆中不再检出HIV RNA。全血组4个时间点,HIV DNA定量结果均未检出;磁珠富集组CD4^(+)T淋巴细胞HIV DNA定量结果分别为:73.4、429.3、137.1、449.9 copies/106个CD4^(+)T细胞。结论磁珠富集法更加灵敏高效检出血液标本中极限低拷贝HIV DNA,为HIV WP感染、ART治疗后突破感染提供早期确证依据。

Objective To establish a magnetic bead enrichment strategy for the detection of human immunodeficiency virus deoxyribonucleic acid(HIV DNA)in peripheral blood,and to verify the improvement of the sensitivity of this method for the detection of HIV DNA in HIV infected patients after early antiretrovital treatment(ART).Methods Peripheral whole blood was collected at 4 timepoints in one ART HIV window period(WP)patient.Peripheral blood mononuclear cells(PBMCs)were isolated on a Ficoll gradient.CD4^(+)T lymphocytes were enriched from total PBMCs by negative sorting.HIV DNA concentration in magnetic beads enriched group and whole blood group was detected by HIV DNA detection kit.Results CD4^(+)T cells were isolated by magnetic beads and identified by FCM for purity at(96.4±2.6)%.The viability was(95.9±2.9)%,as demonstrated by trypan blue staining.The person on continued ART treatment in this study had significantly greater reduction in HIV viral load and undetectable HIV plasma RNA at follow up timepoint 4.No HIV DNA was detected in the whole blood group at all 4 timepoints.The quantitative results of HIV DNA in the CD4^(+)T lymphocyte group of the magnetic bead enrichment group were 73.4,429.3,137.1,449.9 copies/106 CD4^(+)T cell′s respectively.Conclusion The magnetic bead enrichment method can be more sensitive in detecting the limit low copy HIV DNA in blood samples,and provide early confirmatory data for HIV WP infection and breakthrough infection after ART treatment.