Comparison of the Real-Time PCR Tests for Factor V G1691A and Prothrombin G20210A with PCR-Restriction Fragment Length Polymorphism and Direct Sequencing Tests.
10.15263/jlmqa.2015.37.1.37
- Author:
Hyunjung KIM
1
;
Gun Dong LEE
;
Sang Yoon LEE
;
Woori JANG
;
Joonhong PARK
;
Hyojin CHAE
;
Myungshin KIM
;
Yonggoo KIM
Author Information
1. Department of Laboratory Medicine, The Catholic University of Korea College of Medicine, Seoul, Korea. microkim@catholic.ac.kr
- Publication Type:Original Article
- Keywords:
Real-time polymerase chain reaction;
Factor V G1691A;
Prothrombin G20210A;
Thrombophilic mutation;
Factor V Leiden
- MeSH:
DNA;
Factor V*;
Humans;
Indicators and Reagents;
Limit of Detection;
Polymerase Chain Reaction;
Prothrombin*;
Real-Time Polymerase Chain Reaction*;
Thromboembolism;
World Health Organization
- From:Journal of Laboratory Medicine and Quality Assurance
2015;37(1):37-43
- CountryRepublic of Korea
- Language:Korean
-
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.
