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ARMS TaqMan real‐time PCR for genotyping factor V Leiden mutation in Han Chinese
Author(s) -
Yu Pan,
Dai Yibei,
Dong Jiantao,
Zhang Luyan,
Ping Ying,
Wang Xuchu,
Wang Danhua,
Tao Zhihua
Publication year - 2020
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.202000193
Subject(s) - taqman , genotyping , amplicon , microbiology and biotechnology , genetics , mutation , factor v leiden , biology , factor v , nonsense mutation , polymerase chain reaction , missense mutation , gene , genotype , medicine , venous thrombosis , surgery , thrombosis
Factor V Leiden (FV Leiden ) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR‐based methods for detecting FV Leiden mutation; however, these methods have disadvantages such as time‐consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time‐saving, accurate, and gel‐free method, called amplification refractory mutation system (ARMS) TaqMan real‐time PCR, for detecting FV Leiden mutation. We severally designed two specific reverse primers for mutant and wild‐type through intentional introduction of mismatched nucleotide at the penultimate 3′ position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan‐probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild‐type and one heterozygote. Afterward, 50 randomly picked wild‐type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as “Gold standard method.” Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FV Leiden was calculated the in Han Chinese. Given the above results, A FV Leiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real‐time PCR is an ideal detecting system for genotyping FV Leiden mutation in clinical application, and FV Leiden mutation exists in Han Chinese despite extremely low prevalence.