基于数字PCR技术的Y染色体微缺失检测方法开发  

Development of Y Chromosome Microdeletion Detection Method Based on Digital PCR Technology

作　　者：袁通阔 张红国 尹焕才[2] 刘睿智[3] 殷建[2] YUAN Tong-kuo;ZHANG Hong-guo;YIN Huan-cai;LIU Rui-zhi;YIN Jian(College of Life Sciences, Shanghai University, Shanghai 200444, China;Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China;Bethune First Hospital of Jilin University, Changchun 130021, China)

机构地区：[1]上海大学生命科学学院,中国上海200444 [2]中国科学院苏州生物医学工程技术研究所,中国苏州215163 [3]吉林大学白求恩第一医院,中国长春130021

出　　处：《湖南师范大学自然科学学报》2021年第1期47-55,共9页Journal of Natural Science of Hunan Normal University

基　　金：国家科技重大专项(2017ZX10302301-003);国家自然科学基金项目(21876198);中国科学院苏州生物医学工程技术研究所自主部署项目(Y95P061P05)。

摘　　要：为验证数字PCR(dPCR)技术在Y染色体微缺失检测中应用的可行性,以无精子区域(Azoospermia factor,AZF)中的AZFa,AZFb及AZFc为靶标,选择特异性引物并验证其有效性,通过退火温度优化去除可能的非特异性扩增,最终通过已知缺失类型的临床样本验证检测方法的灵敏度及准确性。优化后的dPCR方法检测样本的浓度可低至0.1 mg·L^(-1),该值远低于常规qPCR的检测限(1 mg·L^(-1)),并能够准确反映Y染色体的缺失状况。因此dPCR可准确反映样本中的Y染色体微缺失状况,可开发成为男性不育症及其他生殖诊断中的重要技术。To verify the feasibility of digital PCR technology in the detection of Y chromosome microdeletions,in this work,we made use of AZFa,AZFb and AZFc in the azoospermia factor(AZF)as targets,selected specific primers,and verified their effectiveness.After that,we optimized the annealing temperature to remove possible non-specific amplifications.Finally,the sensitivity and accuracy of the detection method have been verified by clinical samples with known missing types.The optimized digital PCR method can be used to detect sample concentrations as low as 0.1 mg·L^(-1),one magnitude lower than the detection limit of the conventional fluorescent quantitative PCR(qPCR,1 mg·L^(-1)).This new technology can be applied to accurately reflect the Y chromosome deletion status.The digital PCR method established in this work can also be utilized to accurately reflect the Y chromosome microdeletion,and further be developed as an important technology in male infertility and other reproductive diagnoses.

关 键 词：数字PCR qPCR Y染色体微缺失 

分 类 号：R698.2[医药卫生—泌尿科学] R440[医药卫生—外科学]