Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria.
Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria; Medical Central Laboratories, Feldkirch, Austria; Private University of the Principality of Liechtenstein, Triesen, Liechtenstein.
Clin Chim Acta. 2019 Jan;488:221-225. doi: 10.1016/j.cca.2018.11.016. Epub 2018 Nov 12.
Practically, the initial step of genetic analysis is the extraction of DNA from blood or other cells, which is often time consuming and cost-intensive. We aimed at establishing a real-time PCR protocol for the detection of the venous thromboembolism associated mutations factor V Leiden (F5 c.1691G>A; p.R506Q) and prothrombin (F2) c.20210G>A from whole blood, without DNA extraction.
F5 c.1691G>A (p.R506Q) and F2 c.20210G>A mutations were determined in 205 EDTA anti-coagulated whole blood samples from patients who underwent routine clinical genotyping using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with in-house developed TaqMan primer-probe assays.
Validity score values of genotype calls using whole blood were similar and did not significantly differ compared to those using genomic DNA as substrate in PCR. Mutation analysis of 205 whole blood samples showed a negligible PCR dropout rate (one in 410 reactions) and were in 100% concordance with results obtained by conventional genotyping.
We successfully established a robust and valid real-time PCR protocol for the detection of the venous thromboembolism associated mutations F5 c.1691G>A (p.R506Q) and F2 c.20210G>A directly from whole blood.
实际上，基因分析的初始步骤是从血液或其他细胞中提取 DNA，这通常既耗时又昂贵。我们旨在建立一种实时 PCR 方案，用于直接从全血中检测静脉血栓栓塞相关突变因子 V 莱顿（F5 c.1691G>A；p.R506Q）和凝血酶原（F2）c.20210G>A，而无需提取 DNA。
使用 DirectBlood Genotyping PCR 试剂盒（德国康斯坦茨的 myPOLS Biotec）和内部开发的 TaqMan 引物探针检测法，在 205 例接受常规临床基因分型的 EDTA 抗凝血全血样本中确定 F5 c.1691G>A（p.R506Q）和 F2 c.20210G>A 突变。
使用全血进行基因分型的有效性评分值与使用基因组 DNA 作为 PCR 底物的评分值相似，且无显著差异。对 205 份全血样本的突变分析显示，PCR 缺失率可忽略不计（410 次反应中只有 1 次），与传统基因分型的结果完全一致。
我们成功建立了一种稳健且有效的实时 PCR 方案，用于直接从全血中检测静脉血栓栓塞相关突变 F5 c.1691G>A（p.R506Q）和 F2 c.20210G>A。
