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Clin Chim Acta. 2019 Jan;488:221-225. doi: 10.1016/j.cca.2018.11.016. Epub 2018 Nov 12.

Direct blood PCR: TaqMan-probe based detection of the venous thromboembolism associated mutations factor V Leiden and prothrombin c.20210G>A without DNA extraction.

Author information

1
Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria.
2
Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria; Medical Central Laboratories, Feldkirch, Austria; Private University of the Principality of Liechtenstein, Triesen, Liechtenstein.
3
Medical Central Laboratories, Feldkirch, Austria.
4
Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria; Private University of the Principality of Liechtenstein, Triesen, Liechtenstein; Division of Angiology, Swiss Cardiovascular Center, University Hospital of Berne, Berne, Switzerland; Drexel University College of Medicine, Philadelphia, PA, USA.
5
Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT), Feldkirch, Austria. Electronic address: axel.muendlein@vivit.at.

Abstract

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

PMID:
30439355
DOI:
10.1016/j.cca.2018.11.016
[Indexed for MEDLINE]
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