微滴式数字PCR(ddPCR)快速检测α珠蛋白基因ααα^(anti-3.7)三联体  

Rapid detection of alpha-globin gene ααα^(anti-3.7) triplets with droplet digital PCR

作　　者：龚小倩 杨学煌 乔伶俐 陈亚军 周万军 GONG Xiaoqian YANG Xuehuang QIAO Linli CHENG Yajun ZHOU Wanjun(Shaoguan Maternal and Child Healthcare Hospital, Shaoguan 512026, China Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China)

机构地区：[1]广东省韶关市妇幼保健院,广东韶关512026 [2]南方医科大学基础医学院医学遗传学教研室,广东广州510515

出　　处：《南方医科大学学报》2017年第9期1265-1269,共5页Journal of Southern Medical University

基　　金：韶关市科技计划项目(2015CX/K19)

摘　　要：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Taq Man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。Objective To establish a rapid method for detection of alpha-globin gene ααα^anti-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 Taq Man 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-3.7from the 60 clinical samples,and the results were verified by MLPA.The sensitivity and accuracy of this method were both100% for detecting alpha-globin gene ααα^anti-3.7.Conclusion This ddPCR-based method for detecting ααα^anti-3.7 triplet can be applied for population screening and in routine clinical molecular diagnosis with simple operation,rapid analysis and accurate results.

关 键 词：数字PCR 珠蛋白基因 多拷贝 地中海贫血 

分 类 号：R440[医药卫生—诊断学] R556.61[医药卫生—临床医学]