Objective: To establish a rapid method for detection of alpha-globin gene αααanti-3.7 based on droplet digital PCR (ddPCR) technique.
Methods: The differential sequence between the X1 and Y1 box of α1 gene was selected as the amplicon of the target gene with β-actin as the reference gene. The specific primers and TaqMan probes were designed, and then a quantitative method for detecting the copy number was established based on ddPCR technique. The sensitivity and accuracy of the method were evaluated by detecting 28 samples of known genotypes and 60 clinical samples.
Results: The ddPCR-based method accurately identified the genotypes of all the 28 samples with known genotypes and detected 5 cases of αα/αααanti-3.7 from the 60 clinical samples, and the results were verified by MLPA. The sensitivity and accuracy of this method were both 100% for detecting alpha-globin gene αααanti-3.7.
Conclusion: This ddPCR-based method for detecting αααanti-3.7 triplet can be applied for population screening and in routine clinical molecular diagnosis with simple operation, rapid analysis and accurate results.
目的: 建立一种基于微滴式数字PCR（ddPCR）技术的α珠蛋白基因αααanti-3.7快速检测方法。
方法: 以β-actin为参比基因、α1基因X1/Y1盒之间的差异性序列为目的基因代表性扩增子，设计特异性引物和TaqMan探针，建立基于ddPCR技术的拷贝数定量方法；检测28例已知基因型和60例临床样本，评价此方法的灵敏度和准确性。
结果: 采用本研究建立的方法，28例已知基因型样本的检测结果均与靶基因型结果相符；60例临床样本中检出5例αα/αααanti-3.7，与MLPA的验证结果一致；方法学评价结果显示此ddPCR体系的灵敏度与准确性均达100%。
结论: 建立了一种α珠蛋白基因αααanti-3.7三联体ddPCR检测方法，操作简单快速、结果准确可靠，可应用于人群筛查和临床常规分子诊断。