J Lab Med Qual Assur.  2015 Mar;37(1):37-43. 10.15263/jlmqa.2015.37.1.37.

Comparison of the Real-Time PCR Tests for Factor V G1691A and Prothrombin G20210A with PCR-Restriction Fragment Length Polymorphism and Direct Sequencing Tests

• Kim H
• Lee GD
• Lee SY
• Jang W
• Park J
• Chae H
• Kim M
• Kim Y

• KMID: 1789361
• DOI: http://doi.org/10.15263/jlmqa.2015.37.1.37

Abstract

BACKGROUND
Factor V (FV) G1691A and prothrombin G20210A mutations are the most common targets of genetic tests for thromboembolism. This study compared the ability of real-time PCR to detect FV G1691A and prothrombin G20210A (BioSewoom, Korea) with that of PCR-restriction fragment length polymorphism (RFLP) and direct sequencing, to evaluate diagnostic equivalency.
METHODS
Real-time PCR was compared with PCR-restriction fragment length polymorphism (RFLP) and direct sequencing using patients' samples as well as heterozygous and homozygous World Health Organization (WHO) reference reagent DNA. The limit of detection (LoD) for real-time PCR was determined using WHO reference reagents.
RESULTS
All 141 and 156 patient samples were tested for the FV G1691A and prothrombin G20210A mutations, respectively; the results from all three methods (real-time PCR, PCR-RFLP, and direct sequencing) consistently showed that the samples were wild type. Each of the three methods showed the same results in tests using heterozygous and homozygous DNA from the WHO reference reagents. The LoD of wild type and homozygous samples was 65.16 pg/mL for FV G1691A, and 61.3 pg/mL for prothrombin G20210A. The LoD of heterozygous samples was 1,650.0 pg/mL for FV G1691A and 1,640.0 pg/mL for prothrombin G20210A.
CONCLUSIONS
The real-time PCR test kits for FV G1691A and prothrombin G20210A showed reliable equivalency with PCR-RFLP and direct sequencing, and could be useful tests to detect gene polymorphisms for thromboembolism.

Keyword

Real-time polymerase chain reaction; Factor V G1691A; Prothrombin G20210A; Thrombophilic mutation; Factor V Leiden

MeSH Terms

DNA
Factor V*
Humans
Indicators and Reagents
Limit of Detection
Polymerase Chain Reaction
Prothrombin*
Real-Time Polymerase Chain Reaction*
Thromboembolism
World Health Organization
DNA
Factor V
Indicators and Reagents
Prothrombin