液态芯片技术检测Y 染色体微缺失方法的优化

目的优化基于液态芯片检测高通量、快检测Y染色体微缺失的方法。方法以sY84、sY86（AZFa）,sY127、sY134（AZFb）,sY152、sY153（AZFd）,sY242、sY254、sY255（AZFc）等9个Y染色体微缺失的STS位点作为检测区域,以sY14（SRY,男性性别决定基因）位点作内部对照。优化探针引物组合,建立基于液态芯片的多重PCR-技术检测Y染色体微缺失的方法。结果优化的引物对在多重PCR体系中显示特异的扩增效率;多重PCR产物与STS特异的探针微球杂交后,在Luminex上显示非常强的特异性的荧光信号。结论多重PCR-液态芯片技术检测Y染色体微缺失的方法具有高灵敏性、高特异性,高准确性,操作简单,结果可靠,使快速、高通量筛选男性不育症的分子缺陷成为可能。

Optimized method for the detection of Y chromosome microdeletions by liquid gene chip

Objective To optimize a rapid,accurate and high-throughput method for the detection of Y chromosome microdeletions.Methods Nine sequence tagged site(STSs) such as sY84,sY86(AZFa);sY127,sY134(AZFb);sY152,sY153(AZFd);sY242,sY254,sY255(AZFc) were selected to detect Y chromosome microdeletions and sY14(SRY,sex-determining region on Y gene) as internal control.Shorter and more effective primers were designed according to STSs'sequences.Probes targeting the specific STS were tested by reverse dot hybridization.The determined combination of primer and probes was employed in liquichip-based multiplex PCR system for detection of chromosome Y microdeletion.Results The optimized primer combinations in multiplex PCR system gave a superior amplification efficiency and specificity.After PCR products hybridizaed with probe-coupled microsphere,Luminex showed distinctive fluorescent signal for individual probes.Conclusion The multiplex PCR combining liquidchip technique for the detection of Y chromosome microdeletions is very sensitive,highly specific,accurate and reliable.It will facilitate the high-throughput screen the molecular defects for azoospermia.