Holodniy M, Kim S, Katzenstein D, Konrad M, Groves E, Merigan T C
Center for AIDS Research, Stanford University Medical Center, California 94305.
J Clin Microbiol. 1991 Apr;29(4):676-9. doi: 10.1128/jcm.29.4.676-679.1991.
Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.
病毒特异性序列的基因扩增作为一种检测或确认人类免疫缺陷病毒(HIV)感染的方法被广泛应用。在本研究中,我们使用酶联亲和测定法对来自全血、血浆以及在四种常用抗凝剂(酸性枸橼酸盐葡萄糖、乙二胺四乙酸钠、草酸钾和肝素钠)存在的情况下收集的分离单核细胞的聚合酶链反应产物进行定量。在对从肝素钠抗凝的标本中提取的全血、洗涤后的单核细胞和血浆进行核酸扩增后,观察到产物信号衰减。这些对基因扩增的抑制作用可用肝素酶逆转。仅添加0.05单位的肝素就能完全抑制来自胎盘DNA的HLA - DQa序列的扩增。我们得出结论,肝素可导致基因扩增减弱或抑制。缺乏抑制活性的酸性枸橼酸盐葡萄糖和乙二胺四乙酸钠是预期进行聚合酶链反应扩增时临床血液样本最合适的抗凝剂。