An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cyropreserved plasma
An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cyropreserved plasma
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DOI：
10.1016/j.jviromet.2011.05.012
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发表时间：
2011-09-01
影响因子：
3.1
通讯作者：
Chen, Yue
中科院分区：
文献类型：
--
作者：
Ding, Ming;Bullotta, Arlene;Chen, Yue
肝素钠是一种广泛用于血液采集的抗凝血剂，已知在聚合酶链反应（PCR）检测中可抑制DNA聚合酶活性。然而，在20世纪80年代和90年代初，在多中心艾滋病队列研究中收集的所有冷冻保存的血浆样本都来自肝素处理过的血液，这给PCR测定这些样本中所含的靶核酸带来了问题。在本研究中，优化了一种核酸提取工艺，从提取的核酸中去除肝素。利用优化后的方法，用逆转录酶（RT）实时荧光定量PCR和实时荧光定量PCR检测从乙二胺四乙酸（EDTA）、柠檬酸葡萄糖（ACD）或肝素钠管中加入到正常供者血液中的HIV-1和巨细胞病毒（CMV）载量。在EDTA-收集的HIV-1和CMV感染活动性患者的血液中也检测到类似的HIV-1和CMV载量。ACD或肝素钠处理管RT - real-time和real-time PCR。结果表明，优化后的核酸提取流程有效地消除了肝素对实时PCR性能的抑制作用。该方法可用于从存档的肝素化血浆中提取核酸，用于基于PCR的目标分子定量。(C) 2011 Elsevier B.V.版权所有
Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-.ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules. (C) 2011 Elsevier B.V. All rights reserved.