电化学发光PCR方法检测结肠癌中K-ras癌基因点突变

发展了一种可用于快速检测K-ras癌基因点突变的电化学发光PCR(ECL-PCR)分析方法,该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;随后,采用限制性内切酶MvaI对扩增产物进行酶切,由于突变导致酶切位点的丢失,所以只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到反应池中进行电化学发光检测。采用该法对20例结肠癌组织中的K-ras癌基因第12位密码子进行点突变分析,得出其中有9例存在点突变,点突变率为45%。该方法操作简便、安全、快速、灵敏,可用于快速检测K-ras癌基因点突变。

An ECL-PCR Method for the Detection of K-ras Point Mutation in Colorectal Cancer

An electrochemiluminescence-polymerase chain reaction(ECL-PCR) method for the detection of K-ras point mutation in colorectal cancer(CAC) was developed.Briefly,the target gene was amplified by a Ru(bpy)32 -labeled forward primer and a biotin-labeled reverse primer,and then followed by digestion with a restriction enzyme,MvaI,which only cut the wild-type amplicon containing its cutting site.Digested product was detected by ECL assay after adsorption of the resulting DNA duplex to the solid phase.Twenty CAC samples were analyzed by the ECL-PCR assay.The positive rate of K-ras point mutation was 45 %.The ECL-PCR method is useful in K-ras point mutation detection due to its sensitivity,rapidness,simplicity and safety.