全血直接多重PCR法快速检测Y染色体微缺失  

Direct nmltiplex-PCR from whole blood for rapid detection of Y chronmsome microdeletions

作　　者：卜莹 黄欢 武海萍 张晓丹 周国华 崔英霞[2] 姚兵[2] 芦洪涌[2] 项静英[3] 

机构地区：[1]华东医学生物技术研究所,南京210002 [2]南京大学医学院临床学院金陵医院中心实验科 [3]无锡市妇幼保健院

出　　处：《中华医学遗传学杂志》2008年第4期406-409,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（30270368）

摘　　要：目的以全血为起始模板直接进行PCR多重扩增，建立一种快速、简便的Y染色体微缺失检测方法。方法用作者自行研制的“HpH-Buffer”，以不经基因组DNA提取的抗凝全血为模板直接进行多重PCR扩增。分别在5管中检测无精症因子（azoospermia factor，AZF）区域的a区、b区和c区共12个序列标签位点（sequence tagged sites，SIS）。为保证方法有效性，加做Y染色体性别决定区（sex-determining region Y，SRY）和X／Y连锁锌指蛋白基因（X-linked or Y-linked zinc fingergene，ZFX／Y）作为内控。同时，为考察方法的准确性，对每例血液样本提取基因组DNA平行实验。结果共检测了156例男性血液样本，每组实验均加做阳性对照（正常已生育男性样本）和阴性对照（正常女性样本），以保证实验有效性。结果表明，156例样本中有144例无缺失，AZFa区缺失1例，AZFb区缺失1例，AZFc区缺失7例，AZFb和AZFc区缺失1例，AZFa、AZFb和AZFc区全缺失2例。以全血为模板和以基因组DNA为模板进行扩增所得到的实验结果完全一致。结论所建立的方法省略了DNA提取这一繁琐的步骤，减少了PCR实验过程中可能出现的污染，缩短了操作时间，节约了实验成本。可在2小时内完成所有检测过程，有效地提高了临床检测效率。“HpH-Buffer”的试剂成本很低，全部检测成本与普通PCR扩增基本相同。Objective To establish a rapid and simple method to detect Y chromosome microdeletions directly using whole blood as starting material for muhiplex-PCR. Methods Using a self-prepared ＂HpH-Buffer＂, multiplex- PCR amplification was directly carried out from the anticoagulant whole blood sample without DNA extraction step. Twelve sequence tagged sites （SIS）, namely SY84, SY86, SY127, SY134, SY124, SY132, SY152, SY157, SY239, SY242, SY254 and SY255, in AZFa, AZFb, and AZFc gene regions were detected in 5 different tubes. In order to ensure the validity of the experiments, sex-determining region Y （SRY） and X-linked or Y-linked zinc finger gene （ZFX/ Y） were used as internal controls. Furthermore, conventional PCR using genomic DNA extracted from each blood sample was performed in parallel for evaluating the accuracy of the experiments. Results A total of 156 male samples were detected, and a normal male sample and a female sample were used as a positive and a negative control respectively. The results showed that 144 samples had no deletion; one sample was AZFa-deleted; one sample was AZFb-deleted; seven samples were AZFc-deleted; one sample was both AZFb- and AZFc-deleted; and two samples were all AZFa-, AZFb- and AZFc-deleted. The observed results from two kinds of starting material （whole blood and purified DNA） are completely consistent. Conclusion In our method, PCR amplification was directly carried out from whole blood without any DNA extraction step. So it has the advantages of low cost, simple process, time-saving operation and less cross-contamination. The whole process can be completed within 2 hours. Thus the efficiency of clinical detection is improved greatly.

关 键 词：Y染色体微缺失 无精症因子 多重聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]