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.

中文翻译：

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针对汉族人基因型因子V Leiden突变的ARMS TaqMan实时PCR。

因子V莱顿（FV _Leiden）是FV基因第10外显子的1691位（G1691A）错义突变，是静脉血栓形成的遗传风险。当前，有几种基于PCR的方法可检测FV _Leiden突变。但是，这些方法具有诸如耗时，繁琐的步骤以及潜在的危险凝胶等缺点。本研究的目的是开发一种简单，省时，准确且无凝胶的方法，称为扩增难治性突变系统（ARMS）TaqMan实时PCR，用于检测FV _Leiden突变。我们通过在倒数第二个3'位故意引入错配的核苷酸，分别设计了两种针对突变型和野生型的特异性反向引物。尽管靶扩增子的扩增效率降低，但是另一种相应的扩增子几乎被完全抑制。然后，设计了特定的TaqMan探针来检测目标扩增子。建立的方法用于检测500例汉族人群未选择的样本，结果显示499例野生型和1例杂合子。之后，通过双向DNA测序重新检查了50例随机挑选的野生型病例和1例杂合子，这被认为是“金标准方法”。令人兴奋的是，两种方法检测到的结果是完全一致的。最后，_莱顿病毒的等位基因频率是用汉文计算出来的。根据上述结果，在汉族人的500个随机样本中发现了FV _Leiden杂合子，等位基因频率为0.1％。总之，ARMS TaqMan实时PCR是临床上对FV _Leiden突变进行基因分型的理想检测系统，尽管汉族人FV _Leiden突变的患病率极低，但仍存在。