变性高效液相色谱快速诊断儿童型脊肌萎缩症

目的建立变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)快速诊断儿童型脊肌萎缩症(spinal muscular atrophy,SMA)的新方法.方法 (1) 用二重聚合酶链反应(PCR)扩增运动神经元成活基因(survival motor neuron gene,SMN)外显子7、8及周围部分内含子序列;(2)纯化PCR产物以除去其中的引物、dNTP及缓冲液中的盐粒子等成分;(3)采用多重引物延伸反应特异性检测能将SMN1基因与SMN2基因区分开的3个特异性位点;(4)将延伸反应的产物用DHPLC在完全变性的条件下分析.结果将建立的新方法与传统的PCR-酶切法进行盲法对比试验,检测了30例标本(包含20例SMA患儿和10例正常人群外周血基因组标本)以验证新方法的特异性和可靠性,其诊断结果与PCR-酶切法结果完全一致.结论多重引物延伸反应结合DHPLC分析技术是一种可用于临床SMA基因诊断、产前诊断及胚胎种植前遗传学诊断的高效、灵敏、可靠、快速、简便的新方法.

A Simple and Rapid Method for Spinal Muscular Atrophy Identification by DHPLC

Objective To develop an accurate,reliable ,simple and rapid method to diagnosis spinal muscular(SMA) by denaturing high pe rformance liquid chromatography(DHPLC).Methods (1) Duplex P CR amplifies exon7,8 along with their flanking intron;(2) Quickly purifies the P CR product to remove the excess primers,deoxynucleotiedes and components in buff er;(3) Multiplex primer extension reaction specifically detects three specific a llocations,which can distinguish SMN1 gene from SMN2 gene;(4) The above products on the DHPLC were analyzed under the completely denaturing conditions. Results By a blind analysis,30 samples including SMA patients and n ormal peoples are identified simultaneously by DHPLC and PCR followed by the res triction enzyme digestion method to test the specificity and reliability of the new method.The results of both methods are completely agreed with each other. Conclusion The multiplex primer extension reaction coupled wit h DHPLC technology is a high throughout,sensitive,reliable,simple and quick meth od for diagnosing SMA,and it can be applied in clinic.