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pISSN 2950-9114 eISSN 2950-9122
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Original Article

J Lab Med Qual Assur 2015; 37(1): 37-43

Published online March 31, 2015


Copyright © Korean Association of External Quality Assessment Service.

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

Hyunjung Kim1, Gun Dong Lee1,2, Sang Yoon Lee1,2, Woori Jang1,2, Joonhong Park1,2, Hyojin Chae1,2, Myungshin Kim1,2, and Yonggoo Kim1,2

1Department of Laboratory Medicine and 2Catholic Genetic Laboratory Center, The Catholic University of Korea College of Medicine, Seoul, Korea

Correspondence to:Myungshin Kim
Department of Laboratory Medicine, Seoul St. Mary’s Hospital, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea.
Tel: +82-2-2258-1645
Fax: +82-2-2258-1719
E-mail: microkim@catholic.ac.kr

Received: December 17, 2014; Revised: February 10, 2015; Accepted: February 23, 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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, PCRRFLP, 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. (J Lab Med Qual Assur 2015;37:37-43)

Keywords: Real-time polymerase chain reaction, Factor V G1691A, Prothrombin G20210A, Thrombophilic mutation , Factor V Leiden

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