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目的 建立变性高效液相色谱(denaturinghighperformanceliquidchromatography, DHPLC) 快速诊断儿童型脊肌萎缩症(spinalmuscularatrophy, 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(survivalmotorneurongene, 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。
Objective To establish a new method of rapid diagnosis of spinal muscular atrophy (SMA) by denaturing high performance liquid chromatography (DHPLC). Methods (1) Exon 7 and exon 7 of Survivin neuron survival gene (SMN) were amplified by double polymerase chain reaction (PCR); (2) PCR products were purified to remove Primers, dNTPs and salt particles in the buffer solution; (3) three specific loci that can separate the SMN1 gene and the SMN2 gene by using the multiplex primer extension reaction specificity detection; (4) the product of the extension reaction DHPLC is analyzed under fully denaturing conditions. Results The new method was compared with the traditional PCR-restriction method to compare the specificity and specificity of the new method in 30 specimens (including 20 cases of SMA patients and 10 normal human peripheral blood genomic samples) Reliability, its diagnostic results and PCR-enzyme digestion results exactly the same. Conclusions Multiple primer extension reaction combined with DHPLC is a new efficient, sensitive, reliable, rapid and simple method for clinical diagnosis of SMA, prenatal diagnosis and preimplantation genetic diagnosis.