F8 intron 22 inversion (Inv22) accounts for about 40% of severe hemophilia A (HA) cases and is mainly genotyped by long-distance PCR (LD-PCR) or inverse-PCR (I-PCR). These methods require long separation times or enzymatic digestion. We aimed to shorten the separation time of LD-PCR. Long-read sequencing was applied for LD-PCR products from 20 Inv22 patients and 4 controls to validate the differences between products generated using P-Q and P-B primer pairs in LD-PCR. We then confirmed two unique regions (chrX: 154879481-154880814, chrX: 155376388-155376505, GRCh38) in the PCR products from P-Q and P-B primer pairs, respectively. The nested PCR P1, Q1, and B1 primers were located near the homologous sequence and two unique regions, respectively. The P1-Q1 and P1-B1 primer pairs generated 1621 bp and 540 bp fragments, respectively, and the Inv22 carriers produced both fragments. In total, 228 previously diagnosed subjects including 39 Inv22 carriers, 52 Inv22 patients, 82 Inv22 negative males, and 55 Inv22 negative females were genotyped using nested PCR, and the results revealed excellent sensitivity and specificity (100 and 100%, respectively). The separation time was shortened from 5 to 0.5 h. Therefore, we present a rapid genotyping method for F8 Inv22 by nested PCR based on LD-PCR.
基金:
National Natural Science Foundation of China [81770168] Funding Source: Medline
语种:
外文
被引次数:
WOS:
PubmedID:
中科院(CAS)分区:
出版当年[2019]版:
大类|3 区医学
小类|4 区心脏和心血管系统4 区血液学4 区外周血管病
最新[2025]版:
大类|3 区医学
小类|3 区血液学4 区心脏和心血管系统4 区外周血管病
JCR分区:
出版当年[2018]版:
Q2PERIPHERAL VASCULAR DISEASEQ2HEMATOLOGYQ2CARDIAC & CARDIOVASCULAR SYSTEMS
第一作者单位:[1]Huazhong Univ Sci & Technol, Tongji Hosp, Tongji Med Coll, Dept Lab Med, Wuhan 430030, Peoples R China
通讯作者:
推荐引用方式(GB/T 7714):
Wang Xiong,Hu Weihong,Gao Yong,et al.Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR[J].JOURNAL OF THROMBOSIS AND THROMBOLYSIS.2020,49(4):591-601.doi:10.1007/s11239-020-02043-5.
APA:
Wang, Xiong,Hu, Weihong,Gao, Yong,Li, Dengju&Lu, Yanjun.(2020).Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR.JOURNAL OF THROMBOSIS AND THROMBOLYSIS,49,(4)
MLA:
Wang, Xiong,et al."Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR".JOURNAL OF THROMBOSIS AND THROMBOLYSIS 49..4(2020):591-601