Hourfar Michael K, Michelsen Uwe, Schmidt Michael, Berger Annemarie, Seifried Erhard, Roth W Kurt
Institute of Transfusion Medicine and Immunohaematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany.
Clin Chem. 2005 Jul;51(7):1217-22. doi: 10.1373/clinchem.2005.048603.
Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.
We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.
使用磁性固相的提取方案具有很高的自动化潜力。然而，市售的基于磁珠的检测方法要么缺乏病毒诊断所需的灵敏度，要么成本过高。
我们开发了一种水性化学方法，用于使用普通磁性硅胶珠从血浆样本中提取病毒核酸。核酸在具有促盐析盐的酸性条件下与磁珠结合，并在略碱性pH值下洗脱。该方法在标准移液工作站上实施，可在一批中全自动提取多达48个240μL血浆样本。
该方法的检测限与基于离心柱的QIAamp Viral RNA Mini Kit相当，作为比较方法，后者依赖于离液盐并与硅胶膜结合。HIV-1的95%检测限为每PCR 23.1 IU，丙型肝炎病毒（HCV）为每PCR 10.7 IU。在总共178份HIV-1或HCV阳性血浆样本中证实了其适用于临床常规检测。对于所评估的病毒，该方法的线性（R²）＞0.99。
使用不含有机溶剂的试剂可在常见移液机器人上简单且经济高效地实现该方法的自动化，污染风险低。这种新型提取方法的性能特点使其适用于传染病诊断和病毒载量测定。
