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KMID : 0985420150370010037
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Laboratory Medicine and Quality Assurance
2015 Volume.37 No. 1 p.37 ~ p.43
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Comparison of the Real-Time PCR Tests for Factor V G1691A and Prothrombin G20210A with PCRRestriction Fragment Length Polymorphism and Direct Sequencing Tests
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Kim Hyun-Jung
Lee Gun-Dong
Lee Sang-Yoon
Jang Woo-Ri
Park Joon-Hong
Chae Hyo-Jin
Kim Myung-Shin
Kim Yong-Goo
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Abstract
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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.
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KEYWORD
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Real-time polymerase chain reaction, Factor V G1691A, Prothrombin G20210A, Thrombophilic mutation , Factor V Leiden
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