四重TaqMan实时荧光PCR检测α地中海贫血——^(SEA)、—α^(3.7)和—α^(4.2)缺失

Detection of α-thalassemia——^(SEA)、-α^(3.7) and -α^(4.2) deletion by quadruple TaqMan real-time fluorescence PCR

目的建立并评价一种基于珠蛋白基因拷贝数相对定量技术的α地中海贫血(α地贫)--SEA、-α3.7和-α4.2缺失基因型快速检测方法。方法建立四重TaqMan实时荧光PCR体系结合2-ΔΔCt数据分析的方法,同时检测ψα、α2和α1基因相对拷贝数,快速诊断α地贫缺失基因型;以此方法检测28例已知α地贫缺失基因型标本和309例临床标本,并与gap-PCR平行对比检测,评价该方法的准确性与实用性。结果建立的检测体系扩增效率均接近100%;28例已知基因型及309例临床标本的检测结果显示其准确性达到100%,且能有效消除微量或少量外源污染导致的假阴性和假阳性。结论本研究建立的基于珠蛋白基因拷贝数相对定量技术的α地贫--SEA、-α3.7和-α4.2缺失基因型检测方法,操作简单快速,结果准确可靠,适合大规模人群筛查和常规分子诊断。

Objective To establish and evaluate a globin gene copies relative quantitation method for the rapid detection of α-thalasse-^SEA、-α^3.7 and -α^4.2 genotypes. Methods The quadruple TaqMan real-time fluorescence PCR combined with 2 -△△ct, data analysis was established to simultaneously detect the relative copies of ψα, α2 and α1 genes for the rapid diagnosis of deletion genotypes of α- thalassemia. Then, 28 samples with known deletions of α-thalassemia genotypes and 309 clinical samples were detected by the established method, and the obtained results were compared with those from gap-PCR to evaluate the accuracy and practicality of the method. Results The amplification efficiency of the established detection system was close to 100%. The accuracy of the detection system for the results of 28 samples with known deletions of α-thalassemia genotypes and 309 clinical samples was up to 100%. Moreover, the false negative and false positive caused by a small amount or trace of foreign contamination could be effectively eliminated. Conclusion The established detection system based on globin gene copies relative quantitation technique could detect the deletions of α-thalasse- mia -^SEA、-α^3.7 and -α^4.2 genotypes simply, rapidly and accurately, which was suitable for the large-scale population screening and routine molecular diagnosis.