电化学发光-聚合酶链式反应方法检测胰腺癌中K-ras基因点突变

Detection of K-ras Point Mutation in Pancreatic Cancer by Electrochemiluminescence-Polymerase Chain Reaction

发展了一种可用于快速检测胰腺癌中K-ras癌基因点突变的电化学发光-聚合酶链式反应(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;再采用限制性内切酶MvaI对扩增产物进行酶切。由于野生型样品和突变型样品间存在酶切位点的变化,其中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片段收集到检测池中,进行电化学发光检测。采用该法对13例胰腺癌组织中的K-ras癌基因第12位密码子进行点突变分析,只需要10μL样品、20min孵育时间和30s采集时间,就可得出其中有12例存在点突变,点突变率为92.3%。本方法操作简便、安全、快速、灵敏,可用于检测任何一种导致限制性内切酶位点改变的基因点突变。

An electrochemiluminescence polymerase chain reaction （ECL-PCR） method for rapid detection of K-ras point mutation was developed. Briefly, the target gene was amplified by a Ru （bpy）3^2 ＋ -labeled forward primer and a biotin-labeled reverse primer, and then followed by digestion with the restriction enzyme, MvaI, which only cut the wild-type amplicon containing its cutting site. Digestion product was detected by ECL assay after adsorption of the resulting DNA duplex to the solid phase. Thirteen pancreatic cancer samples were analyzed by ECL-PCR assay. The positive rate of K-ras point mutation was 92.3%. The ECL-PCR method is useful in point mutation detection due to its sensitivity, rapidness, simplicity and safety. It can be used to detect a point mutation that creates or destroys a restriction site in any gene.