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Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR

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单位: [1]Huazhong Univ Sci & Technol, Tongji Hosp, Tongji Med Coll, Dept Lab Med, Wuhan 430030, Peoples R China [2]Grand Biosci, Beijing 102206, Peoples R China [3]Huazhong Univ Sci & Technol, Tongji Hosp, Tongji Med Coll, Dept Hematol, Wuhan 430030, Peoples R China
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关键词: Hemophilia A F8 intron 22 inversion Long-distance PCR Long-read sequencing Nested PCR

摘要:
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.

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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
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出版当年[2018]版:
Q2 PERIPHERAL VASCULAR DISEASE Q2 HEMATOLOGY Q2 CARDIAC & CARDIOVASCULAR SYSTEMS
最新[2024]版:
Q2 CARDIAC & CARDIOVASCULAR SYSTEMS Q3 HEMATOLOGY Q3 PERIPHERAL VASCULAR DISEASE

影响因子: 最新[2024版] 最新五年平均 出版当年[2018版] 出版当年五年平均 出版前一年[2017版] 出版后一年[2019版]

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第一作者单位: [1]Huazhong Univ Sci & Technol, Tongji Hosp, Tongji Med Coll, Dept Lab Med, Wuhan 430030, Peoples R China
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