新改良PCR／LDR／毛细管电泳技术产前诊断胎儿β-地中海贫血的可行性研究  

Feasibility of applying the new improved PCR/LDR/capillary electrophoresis on prenatal diagnosis of fetal beta thalassaemia

作　　者：廖茜[1] 易萍[1] 郑英如[1] 俞丽丽[1] 钟小林[1] 黄寅虎[1] 韩磊[1] 朱玉娟[1] 李力[1] 

机构地区：[1]重庆第三军医大学大坪医院野战外科研究所妇产科中心,400042

出　　处：《解放军医学杂志》2014年第3期197-201,共5页Medical Journal of Chinese People's Liberation Army

基　　金：全军计划生育优生优育技术中心基金(505-1545)~~

摘　　要：目的研究采用新改良聚合酶链反应(PCR)/连接酶检测反应(LDR)/毛细管电泳技术产前诊断胎儿β-地中海贫血的可行性。方法使用不同浓度梯度的正常DNA与微量β-地中海贫血突变杂合子DNA混合后进行PCR扩增,扩增产物进行LDR反应后通过毛细管电泳技术检测其连接产物。采用CEQ8000型遗传分析仪分析LDR产物,并观察其灵敏度。结果将含有相同浓度CD17(A→T)突变的扩增产物作为LDR模板,采用8U DNA连接酶进行连接反应,检测灵敏度可达1:5000,产物峰的面积随着正常人外周血DNA浓度的增加而减少,至1:10 000处不能与杂峰区分,且无CD17(A→T)突变扩增产物的阴性对照未检测到LDR产物。正常孕妇血浆DNA中加入20pg CD17(A→T)突变杂合子外周血DNA扩增产物,LDR产物峰可明显与杂峰区分。结论新改良PCR/LDR/毛细管电泳技术有望通过孕妇血浆DNA检测进行β-地中海贫血的产前诊断。Objective To investigate the feasibility of applying new improved polymerase chain reaction （PCR）/ligase detection reaction （LDR）/capillary electrophoresis to the prenatal diagnosis of fetalβ thalassemia from maternal plasma. Methods The mixture of different concentrations of normal DNA and trace β-thalassemia mutation heterozygous DNA were used for PCR, and the amplification products obtained thereafter were used for LDR. The Iigation product was detected by capillary electrophoresis to demonstrate the sensitivity. Results The amplification products containing same concentration of CD17 （A-T） mutation were used as LDR template, 8U DNA ligase reaction was performed. LDR products were analyzed by genetic analyzer CEQ8000 type electrophoresis with a detection sensitivity of 1：5000. Product peak area decreased with the increase of normal peripheral blood DNA concentration gradient. When the concentration reached to 1：10 000, it would be hard to distinguish the product peak and miscellaneous peak. The LDR product was not detected in the product of negative control without CD 17 （A -T） mutation. The LDR results of normal maternal plasma DNA added 20pg CD17 （A-T） heterozygous mutation in peripheral blood DNA amplification products revealed that the product peaks could be clearly distinguished from miscellaneous peaks. Conclusion The new improved PCR/LDR/capillary electrophoresis technology is expected to be used in plasma DNA tests to prenatal diagnosis of fetal beta thalassaemia in pregnant women.

关 键 词：Β地中海贫血 产前诊断 电泳 毛细管 

分 类 号：R715.5[医药卫生—妇产科学]