Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Keywords: Direct blood PCR; Factor V Leiden; Mutation detection; Prothrombin c.20210G>A; TaqMan-probe; Venous thromboembolism.
Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.