多重PCR-毛细管电泳法检测Y染色体微缺失

目的:对多重PCR-毛细管电泳法检测Y染色体微缺失进行评价。方法:选择Y染色体无精子因子(AZF)区域的15个序列标签位点(STS),设计特异性的引物,分成4个不同的多重PCR体系。首先提取临床外周全血样本的基因组DNA。将样本的DNA分别加入到4个不同的PCR体系中进行扩增。将获得的目的DNA片段进行毛细管电泳,并根据预设的AZF marker计算出各检测位点的碱基长度,然后分析临床样本的Y染色体微缺失情况。结果:该方法能够准确检测出临床样本的Y染色体AZF a区域缺失、AZF b区域缺失和AZF c区域缺失等缺失;检测限约为3 ng·反应^-1;正常男性临床样本的检测结果是均未发生Y染色体微缺失;在210例无精子症和少精子症患者中,该方法能够准确检测出19例AZF区域微缺失,包括AZF a区域缺失1例,AZF b区域缺失2例,AZF c区域缺失13例,AZF bc区域缺失1例和AZF abc区域缺失2例。结论:多重PCR-毛细管电泳法具有很好的临床适用性,在临床上可以用于Y染色体微缺失检测,为临床遗传咨询提供建议。

Objective:To evaluate the multiplex polymerase chain reaction(PCR)-capillary electrophoresis method for detecting Y-chromosomal microdeletions.Methods:According to the fifteen sequence tagged sites of Azoospermia factor(AZF) regions on the Y chromosome,the specific primers were designed and divided into four multiple PCR amplification systems.Genomic DNA was extracted from whole blood samples,and respectively amplified by four multiplex PCR amplification systems.Then the target DNA fragments were put in capillary electrophoresis.According to the preset AZF marker,the bases of each locus were calculated and the Y-chromosomal microdeletions were analyzed.Results:The method be used to accurately detect the deletions of AZF region in the clinical samples,such as AZF a deletions,AZF b deletions and AZF c deletions.The limit can reach to 3 ng·action^-1.The results of normal male clinical samples are not the present of Y-chromosomal microdeletions.Among the 210 azoospermia and oligozoospermia patients,19 were found to have Y chromosome microdeletions in AZF regions by this method.Including 1 deletion in AZF a,2 deletions in AZF b,13 deletions in AZF c and 1 deletions in AZF b+AZF c,2 deletions in AZF a+AZF b+AZF c.Conclusion:These findings suggest that the multiplex polymerase chain reaction(PCR)-capillary electrophoresis is accessible for clinical application,and suitable for detecting Y-chromosomal microdeletions in clinical diagnosis and offering advice for genetic counseling.