Development and clinical application of a rapid gene diagnosis method for Thai type thalassemia
-
摘要: 目的:建立一种快速、简便且无需核酸提取的泰国型α缺失地中海贫血的快速基因诊断方法及其在产前诊断中应用。方法:设计可以扩增α-珠蛋白基因簇中--THAI等位基因的特征序列引物组,直接以待测样本为模板,不需要核酸提取,进行聚合酶链反应(PCR)直接扩增,扩增产物进行琼脂糖凝胶电泳即可分析结果。结果:成功建立泰国缺失型地中海贫血的快速基因诊断方法,并且直接以全血、羊水、干血斑、脐带血为模板进行扩增的结果与常规跨越断裂点聚合酶链反应(gap-PCR)技术确定基因型完全一致。结论:该法不需要提取核酸,减少操作步骤与实验室污染、节约成本、快速、准确,可作为常规方法用于临床样品的地中海贫血分子基因检测及巴氏水肿胎和缺失型HbH病的产前诊断及泰国型α-地中海贫血的产前诊断新方法。
-
关键词:
- 泰国型α-地中海贫血 /
- 直接PCR /
- 产前诊断 /
- 基因诊断
Abstract: Objective: To establish a rapid, simple and non nucleic acid extraction based gene diagnosis method for Thai type Thalassemia and its application in prenatal diagnosis.Methods: The characteristic sequence Primers of-thai Allele in-globin gene cluster were designed, and the samples were directly amplified by PCR without nucleic acid extraction, the amplified products were analyzed by Agarose Gel electrophoresis.Results: A rapid genotyping method for Thai type Thalassemia was successfully established, and the results of direct amplification using whole blood, amniotic fluid, DBS and umbilical cord blood were identical with those determined by gap PCR.Conclusion: This method does not need to extract nucleic acid, reduces the operating procedure and the laboratory pollution, saves the cost, is fast, is accurate, thalassemia gene detection as a routine method for clinical samples and prenatal diagnosis of Bart's edematous foetus and absent type HBH are expected to be new methods for clinical prenatal diagnosis of Thai type thalassemia.-
Key words:
- thai type thalassemia /
- direct PCR /
- prenatal diagnosis /
- genetic diagnosis
-
[1] Taher AT,Weatherall DJ,Cappellini MD.Thalassaemia[J].Lancet,2018,391(10116):155-167.
[2] Weatherall DJ.The Evolving Spectrum of the Epidemiology of Thalassemia[J].Hematol Oncol Clin North Am,2018,32(2):165-175.
[3] Saboor M,Mobarki AA,Hamali HA,et al.Frequency and genotyping of alpha thalassemia in individuals undergoing premarital screening[J].J Pak Med Assoc,2021,71(1):101-104.
[4] Galanello R,Cao A.Gene test review.Alpha-thalassemia[J].Genet Med,2011,13(2):83-88.
[5] Barnett R.Thalassaemia[J].Lancet,2019,394(10204):1135.
[6] Mankhemthong K,Phusua A,Suanta S,et al.Molecular characteristics of thalassemia and hemoglobin variants in prenatal diagnosis program in northern Thailand[J].Int J Hematol,2019,110(4):474-481.
[7] Nopparatana C,Nopparatana C,Saechan V,et al.Prenatal diagnosis of α-and β-thalassemias in southern Thailand[J].Int J Hematol,2020,111(2):284-292.
[8] Schrader C,Schielke A,Ellerbroek L,et al.PCR inhibitors-occurrence,properties and removal[J].J Appl Microbiol,2012,113(5):1014-1026.
[9] Bibi S,Siddiqui TR,Alam SE,et al.Comparison of dried blood spots with conventional blood sampling for diagnosis of hepatitis b & c through serological and molecular technique;a pilot study[J].J Pak Med Assoc,2020,70(7):1214-1219.
[10] Sahiratmadja E,Seu M,Nainggolan IM,et al.Challenges in Thalassemia Carrier Detection in a Low Resource Setting Area of Eastern Indonesia:the Use of Erythrocyte Indices[J].Mediterr J Hematol Infect Dis,2021,13(1):e2021003.
[11] Lu F,Dai Q,Zhang X,et al.Comparison between capillary zone electrophoresis and capillary isoelectric focusing for thalassemia screening in southern China[J].J Clin Lab Anal,2018,32(8):e22567.
[12] Al-Soud WA,Jönsson LJ,Râdström P.Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR[J].J Clin Microbiol,2000,38(1):345-350.
[13] Li Y,Pan X,Roberts ML,et al.Stability of global methylation profiles of whole blood and extracted DNA under different storage durations and conditions[J].Epigenomics,2018,10(6):797-811.
[14] Abu Al-Soud W,Rådström P.Effects of amplification facilitators on diagnostic PCR in the presence of blood,feces,and meat[J].J Clin Microbiol,2000,38(12):4463-4470.
[15] 沈茹,陈云明,杨晓红,等.α地中海贫血筛查及诊断技术的进展[J].分子诊断与治疗杂志,2019,11(1):68-72.
[16] Brancaleoni V,Di Pierro E,Motta I,et al.Laboratory diagnosis of thalassemia[J].Int J Lab Hematol,2016,38 Suppl 1:32-40.
[17] Al-Soud WA,Rådström P.Purification and characterization of PCR-inhibitory components in blood cells[J].J Clin Microbiol,2001,39(2):485-493.
[18] Geiger K,Zach C,Leiherer A,et al.Real-time PCR based HLA-B*27 screening directly in whole blood[J].HLA,2020,95(3):189-195.
[19] Grabias B,Essuman E,Quakyi IA,et al.Sensitive real-time PCR detection of Plasmodium falciparum parasites in whole blood by erythrocyte membrane protein 1 gene amplification[J].Malar J,2019,18(1):116.
[20] Wichian P,Yamsri S,Sanchaisuriya K,et al.Whole Blood PCR for Rapid Screening of α0-Thalassemia[J].Ann Clin Lab Sci,2018,48(2):231-235.
[21] Kuehn BM.Dried Blood Spots May Offer Route to Wider Antibody Testing[J].JAMA,2020,324(19):1933.
[22] Cho HD,Kim J,Lee JY,et al.A novel dried blood spots analysis combined with on-spot reaction for determination of trimethylamine N-oxide and its related compounds[J].Talanta,2020,210:120639.
[23] Nittayaboon K,Nopparatana C.Molecular characterization of Hb H disease in southern Thailand[J].Int J Hematol,2018,108(4):384-389.
计量
- 文章访问数: 352
- PDF下载数: 516
- 施引文献: 0