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目的建立一种快速毛细管电泳检测Y染色体AZF区域微缺失诊断男性不育症的方法。方法利用聚乙烯吡咯烷酮(PVP)作为筛分介质及其兼有的“动态”涂渍作用,采用毛细管非涂渍无胶筛分电泳与多重聚合酶链反应(PCR)相结合技术,配以高灵敏度的激光诱导荧光检测器,对Y染色体AZF(azoospermiafactor)区域微缺失病人15个序列标签位点进行直接基因诊断。结果检测38例严重少精或无精症患者,发现10例存在缺失,缺失率为26.3%。对15例已婚有子女的正常人进行了对照检测,其中缺失率为0%,经χ2检验P<0.01,提示严重少精和无精与正常对照组的AZF区域微缺失率差异有统计学意义,明显高于正常对照组。同时,对AZF四个亚区STS微缺失率进行比较,分别将10例AZF微缺失群体中确定AZFc区的缺失率与另三个区域的缺失率进行分别统计分析,发现AZFc占63.4%,AZFa占4.0%,AZFb占15.2%,AZFd占17.4%,χ2检验P<0.05,提示AZFc区的缺失率与另三个区域差异有统计学意义,AZFc区缺失率明显高于AZFa、AZFb及AZFd区。结论毛细管非涂渍无胶筛分电泳与传统的凝胶琼脂糖电泳相比,本方法在分离速度、分辨率及实验成本方面均有较大改善,其与多重PCR相结合使对严重少精和无精症的诊断更加准确、可靠。
OBJECTIVE To establish a rapid capillary electrophoresis method for the detection of male infertility in AZF region of Y chromosome. Methods Polyvinylpyrrolidone (PVP) was used as screening medium and its “dynamic” coating effect. Capillary non-coated gel electrophoresis combined with multiplex polymerase chain reaction (PCR) Sensitivity of laser-induced fluorescence detector, the Y chromosome AZF (azoospermia factor) microdeletions in patients with 15 sequence tag site for direct gene diagnosis. Results 38 patients with severe oligozoospermia or azoospermia were detected and 10 cases were found to be missing, the deletion rate was 26.3%. 15 cases of married children with normal controls were detected, of which the missing rate of 0%, by χ2 test P <0.01, suggesting that severe oligozoospermia and normal control group AZF regional microdeletion rate differences were statistically significant Significance, significantly higher than the normal control group. At the same time, comparing the deletion rate of STS in four sub-regions of AZF, respectively, the deletion rate of AZFc region and deletion rate of the other three regions of 10 AZF micro-deletion groups were statistically analyzed respectively. The results showed that AZFc accounted for 63.4%, AZFa AZFb accounted for 4.0%, AZFb accounted for 15.2%, AZFd accounted for 17.4%, χ2 test P <0.05, suggesting that AZFc deletion rate and the other three differences were statistically significant, AZFc deletion rate was significantly higher than AZFa, AZFb and AZFd . Conclusion Compared with traditional gel agarose gel electrophoresis, this method has a great improvement in separation speed, resolution and experimental cost. Combined with multiplex PCR, And the diagnosis of azoospermia more accurate and reliable.