Establishment and clinical application of PCR-CTPP method for detection of non-deletion alpha thalassemia point mutations
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摘要: 目的 建立一种基于两对交叉引物PCR技术(PCR with confronting two-pair primers，PCR-CTPP)同时快速检测非缺失型α地中海贫血WS、QS和CS三种突变类型的方法及其在临床中的应用。方法 根据α珠蛋白基因中发生WS、QS和CS突变的区域基因序列，分别针对单个位点设计两对引物即通用外引物和特异性引物，经过聚合酶链反应、电泳鉴别基因型，同时检测非缺失型α地中海贫血。将该方法与PCR反向斑点杂交(PCR-RDB)技术比较，验证其检测准确性。结果 PCR-CTPP方法可用于准确检测非缺失型α地中海贫血的WS、QS和CS突变类型，结果显示与常规非缺失型α地中海贫血PCR-RDB检测方法一致。结论 PCR-CTPP技术检测非缺失型α地中海贫血WS、QS、CS三种突变位点，简单快速、经济省力，可用于非缺失型α地中海贫血的快速筛查和分子流行病学研究，适合在一般实验室推广应用。适用于非缺失型α地中海贫血的人群筛查及婚前或产前筛查，适合各个层次医院应用。
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关键词:
- 非缺失型α地中海贫血 /
- PCR-CTPP法 /
- 点突变
Abstract: Objective To establish a method for the simultaneous rapid detection of three mutation types, WS, QS, and CS, in non-deletion alpha thalassemia based on PCR with confronting two-pair primers(PCR-CTPP) and its application in clinical practice.Methods Based on the gene sequences of the region of the α pearl protein gene where WS, QS, and CS mutations occur, two pairs of primers, universal external primers and specific primers, were designed for a single locus, respectively, and the genotypes were identified by a polymerase chain reaction and electrophoresis to detect the non-deletion type of α thalassemia at the same time. The accuracy of the method was verified by comparing it with PCR-RDB.Results The PCR-CTPP method can be used to accurately detect the WS, QS, and CS mutation types in non-deletion α thalassemia, and the results showed agreement with the conventional PCR-RDB assay for non-deletion α thalassemia.Conclusion PCR-CTPP is a simple, rapid, and cost-effective technique for the detection of WS, QS, and CS mutation sites in non-deficiency α thalassemia, which can be used for rapid screening and molecular epidemiological studies of non-deficiency α thalassemia, and it is suitable for general laboratory applications. It is suitable for population screening for non-deletion alpha-thalassemia and premarital or prenatal screening and is suitable for application in hospitals at all levels.-
Key words:
- non-deletion alpha-thalassemia /
- PCR-CTPP method /
- point mutation
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表 1 各组引物序列表
引物 名称 序列编号 序列 引物组 TF SEQ ID NO.1 CAAGACCTACTTCCCC ACTTC TR SEQ ID NO.2 CCATTGTTGGCACATT CCG WF SEQ ID NO.3 AGTTCACCCCTGCGGT GTAC WR SEQ ID NO.4 AGGAACTTGTCCAGGG AGTCC QF SEQ ID NO.5 GCGGTGCACGCCTCTC T QR SEQ ID NO.6 AGAAGCCAGGAACTT GTGCG CF SEQ ID NO.7 GTGCTGACCTCCAAAT ACCCTT CR SEQ ID NO.8 CACCGAGGCTCCAGCA TG 
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表 2 PCR-CTPP法检测非缺失型α地中海贫血的PCR反应体系及反应条件
WS位点PCR反应体系 QS位点PCR反应体系 CS位点PCR反应体系 PCR反应条件 组分 体积/μL 组分 体积/μL 组分 体积/μL 步骤 温度/℃ 时间 HS Taq 0.1 HS Taq 0.1 HS Taq 0.1 1 95 5 min GC PCR Buffer缓冲液 10.0 GC PCR Buffer缓冲液 10.0 GC PCR Buffer缓冲液 10.0 2 94 30 s TF 0.4 TF 0.4 TF 0.4 3 58 35 s TR 0.4 TR 0.4 TR 0.4 4 72 40 s WF 0.5 QF 0.5 CF 0.5 5 72 5 min WR 0.5 QR 0.5 CR 0.5 6 12 12 s dNTPs (2.5 nmol/L) 1.6 dNTPs (2.5 nmol/L) 1.6 dNTPs (2.5 nmol/L) 1.6 H2O 5.5 H2O 5.5 H2O 5.5 总计 19.0 总计 19.0 总计 19.0
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表 3 MT-PCR-CTPP法单管检测非缺失型α地中海贫血双位点的PCR反应体系及反应条件
WS与CS双位点PCR反应体系 QS与CS双位点PCR反应体系 PCR反应条件 成分 体积/μL 成分 体积/μL 步骤 温度/℃ 时间 HS Taq 0.1 HS Taq 0.1 1 95 5 min GC PCR Buffer缓冲液 10.0 GC PCR Buffer缓冲液 10.0 2 94 30 s TF 0.3 TF 0.3 3 58 35 s TR 0.3 TR 0.3 4 72 40 s WF 0.2 QF 0.2 5 72 5 min WR 0.2 QR 0.2 6 12 12 s CF 0.2 CF 0.2 CR 0.2 CR 0.2 dNTPs(2.5 nmol/L) 1.6 dNTPs(2.5 nmol/L) 1.6 H2O 5.9 H2O 5.9 总计 19.0 总计 19.0
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[1] Kattamis A, Kwiatkowski J, Aydinok Y. Thalassaemia[J]. Lancet, 2022, 399: 2310-2324. doi: 10.1016/S0140-6736(22)00536-0
[2] De Simone G, Quattrocchi A, Mancini B, et al. Thalassemias: from gene to therapy[J]. Mol Aspects Med, 2022, 84: 101028. doi: 10.1016/j.mam.2021.101028
[3] 葛艳芬, 赵越, 刘均如, 等. 地中海贫血患者发生慢性肾病的临床特征与风险评估[J]. 临床血液学杂志, 2023, 36(4): 274-278. https://lcxy.whuhzzs.com/article/doi/10.13201/j.issn.1004-2806.2023.04.011
[4] Piel FB, Weatherall DJ. The α-thalassemias[J]. N Engl J Med, 2014, 371(20): 1908-1916. doi: 10.1056/NEJMra1404415
[5] Harewood J, Azevedo AM. Alpha Thalassemia. 2023 Sep 4[M/OL]//StatPearls[Internet]. Treasure Island(FL): StatPearls Publishing, 2024 Jan-. PMID: 28722856.
[6] 冼嘉嘉, 何健淳, 王燕超, 等. 307例非缺失型α-地贫的基因型及血液学特征分析[J]. 分子诊断与治疗杂志, 2020, 12(6): 720-723. doi: 10.3969/j.issn.1674-6929.2020.06.008
[7] Kalle Kwaifa I, Lai MI, Noor SM. Non-deletional alpha thalassaemia: a review[J]. Orphanet J Rare Dis, 2020, 15(1): 166. doi: 10.1186/s13023-020-01429-1
[8] Motiani A, Sonagra AD. Laboratory Evaluation of Alpha Thalassemia. 2023 Mar 23[M/OL]//StatPearls[Internet]. Treasure Island(FL): StatPearls Publishing, 2024 Jan-. PMID: 36508547.
[9] Hassan S, Bahar R, Johan MF, et al. Next-generation sequencing(NGS)and third-generation sequencing(TGS)for the diagnosis of thalassemia[J]. Diagnostics, 2023, 13(3): 373. doi: 10.3390/diagnostics13030373
[10] 陈华叶, 胡俊杰. 罕见地贫分析和检测研究进展[J]. 分子诊断与治疗杂志, 2023, 15(10): 1663-1666, 1684. doi: 10.3969/j.issn.1674-6929.2023.10.002
[11] 潘美秀, 韦松晓, 林伟健, 等. 血液学指标在4种常见α地中海贫血中筛查价值[J]. 临床血液学杂志, 2022, 8(4): 283-286. https://lcxy.whuhzzs.com/article/doi/10.13201/j.issn.1004-2806.2022.04.012
[12] Sabath DE. The role of molecular diagnostic testing for hemoglobinopathies and thalassemias[J]. Int J Lab Hematol, 2023, 45(Suppl 2): 71-78.
[13] 陈辉, 贾玉艳, 黄粤, 等. 地中海贫血基因治疗进展和现状[J]. 广西医科大学学报, 2024, 41(1): 1-10. https://www.cnki.com.cn/Article/CJFDTOTAL-GXYD202401001.htm
[14] Hamid M, Keikhaei B, Galehdari H, et al. Genotype-phenotype correlation in patients with deletional and nondeletional mutations of Hb H disease in Southwest of Iran[J]. Sci Rep, 2022, 12(1): 4856. doi: 10.1038/s41598-022-08986-4
[15] Vijian D, Wan Ab Rahman WS, Ponnuraj KT, et al. Molecular detection of alpha thalassemia: a review of prevalent techniques[J]. Medeni Med J, 2021, 36(3): 257-269.
[16] Tian Q, Lei YL, Xu LL, et al. First report of nondeletional hb H disease caused by an α2-globin gene mutation: HBA2: c. 184A>T[J]. Hemoglobin, 2021, 45(3): 210-211. doi: 10.1080/03630269.2021.1930553
[17] 葛艳芬, 杨玉静, 刘均如, 等. 毛细管电泳技术在非缺失α-珠蛋白生成障碍性贫血中的应用价值[J]. 中国妇幼保健, 2018, 33(16): 3752-3756. https://www.cnki.com.cn/Article/CJFDTOTAL-ZFYB201816050.htm
[18] 马金花, 黄佩, 吴柳松, 等. 200例地中海贫血第三代测序检测及结果分析[J]. 遵义医科大学学报, 2024, 47(3): 237-245. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYYB202403005.htm
[19] Munkongdee T, Chen P, Winichagoon P, et al. Update in laboratory diagnosis of thalassemia[J]. Front Mol Biosci, 2020, 7: 74.
[20] 张婷婷, 徐湘民. 地中海贫血预防和诊疗技术进展[J]. 广东医学, 2023, 44(10): 1189-1193. https://www.cnki.com.cn/Article/CJFDTOTAL-GAYX202310001.htm
[21] 李巍, 蔡志明, 林坚, 等. ARMS法快速鉴定中国人非缺失型α地中海贫血基因型[J]. 中华血液学杂志, 1999, 20(4): 219-220. https://www.cnki.com.cn/Article/CJFDTOTAL-ZHXY904.024.htm
[22] Suhaimi SA, Zulkipli IN, Ghani H, et al. Applications of next generation sequencing in the screening and diagnosis of thalassemia: a mini-review[J]. Front Pediatr, 2022, 10: 1015769.
[23] Hamajima N. PCR-CTPP: a new genotyping technique in the era of genetic epidemiology[J]. Expert Rev Mol Diagn, 2001, 1(1): 119-123.
[24] Jiang SP, Tong YQ, Zhao R, et al. An improved PCR-CTPP assay for the detection of ADH1B Arg48His polymorphism[J]. J Clin Lab Anal, 2018, 32(2): e22268.
[25] Saad AM, Abdel-Megied AES, Elbaz RA, et al. Genetic variants of APEX1 p. Asp148Glu and XRCC1 p. Gln399Arg with the susceptibility of hepatocellular carcinoma[J]. J Med Virol, 2021, 93(11): 6278-6291.
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