Geiger, Kathrin; Leiherer, Andreas; Brandtner, Eva-Maria; Fraunberger, Peter; Drexel, Heinz; Muendlein, Axel (2019). Direct blood PCR: TaqMan-probe based detection of the venous thromboembolism associated mutations factor V Leiden and prothrombin c.20210G>A without DNA extraction. Clinica chimica acta, 488, pp. 221-225. Elsevier 10.1016/j.cca.2018.11.016
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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.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
04 Faculty of Medicine > Department of Cardiovascular Disorders (DHGE) > Clinic of Angiology |
UniBE Contributor: |
Drexel, Heinz |
Subjects: |
600 Technology > 610 Medicine & health |
ISSN: |
0009-8981 |
Publisher: |
Elsevier |
Language: |
English |
Submitter: |
Isabel Lorenz |
Date Deposited: |
20 May 2019 18:46 |
Last Modified: |
05 Dec 2022 15:27 |
Publisher DOI: |
10.1016/j.cca.2018.11.016 |
PubMed ID: |
30439355 |
Uncontrolled Keywords: |
Direct blood PCR Factor V Leiden Mutation detection Prothrombin c.20210G>A TaqMan-probe Venous thromboembolism |
BORIS DOI: |
10.7892/boris.129395 |
URI: |
https://boris.unibe.ch/id/eprint/129395 |
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