荧光杂交探针实时聚合酶链式反应快速检测甘露聚糖结合凝集素启动子区基因变异方法的建立

目的建立用荧光杂交探针实时聚合酶链式反应（Real-timePCR）检测人甘露聚糖结合凝集素（MBL）启动子区基因变异新型的基因分型法——Tm基因分型法。方法收集30例健康献血员外周抗凝全血,提取基因组DNA。通过LightCycler实时PCR仪描绘扩增的目标DNA片段的熔解曲线,根据熔解温度（Tm）峰值判定待测标本的基因变异类型。最后,用基因测序法检测PCR产物来验证Tm基因分型法的准确性。结果建立了新型的Tm基因分型法,且测定PCR产物的时间仅180min,检测结果与测序法完全吻合。结论Tm基因分型法检测人MBL启动子区基因变异快速、准确,重复性好,具有广泛的临床应用前景。

Establishment of rapid genotyping of polymorphisms in promoter region of mannose-binding lectin gene using real-time PCR with fluorescent hybridisation probes

Objective To develop a new genotyping assay-Tm genotyping assay, using real-time PCR with fluorescence hybridisation probe, for the polymorphisms typing in promoter region of mannosebinding lectin（MBL） gene. Methods Genomic DNA was extracted from peripheral blood of 30 healthy blood donorsl Melting curve of target DNA fragments were drawn using real-time PCR through Light Cycler Instrument inte~ated with specific oligonucleotide probes, and the samples were genotyped according .to the peak of the melting temperature （Tm）. At last, PCR products was examined by gene sequencing to validate Tm genotyping assay. Results Tm genotyping assay was established successfully. A full cycle of Tm genotyping assay takes only 180 minutes of period. The results of Tm genotyping assay was completely consistent with the sequencing results. Conclusions The Tm genotyping assay in detecting the polymorphisms of MBL gene was rapid, accurate and excellently reproducible with promising clinical applications.