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羊水直接PCR法快速诊断脊肌萎缩症的实验研究
引用本文:黄欢,王珏,卢守莲,洪蕾,谢爱萍,曹郡,孙丽洲. 羊水直接PCR法快速诊断脊肌萎缩症的实验研究[J]. 生物磁学, 2013, 0(35): 6845-6847,6903
作者姓名:黄欢  王珏  卢守莲  洪蕾  谢爱萍  曹郡  孙丽洲
作者单位:南京医科大学第一附属医院产科,江苏南京210029
基金项目:国家自然科学基金项目(812v0710,21305069);江苏省妇幼保健重点学科科研项目(FXK201217);江苏省妇幼保健院青年人才培养项目(FRC201302)
摘    要:目的:建立一种快速、简便的产前诊断脊肌萎缩症的检测方法。方法:基于运动神经元生存基因SMNc和SMNt的两个同源拷贝碱基的差异,对SMNt基因第7、8外显子进行缺失分析,抽取孕妇羊水后用作者自行研制的“HpH—Buffer”,以离心后羊水沉淀中的胎儿细胞为模板直接进行PCR扩增,扩增产物进行酶切限制性片断长度多态性分析,对2例有脊肌萎缩症患儿生育史的孕妇怀有的胎儿进行产前基因诊断。同时,为考察羊水直接扩增方法的效率,对羊水样本直接扩增和对羊水样本中提取的基因组DNA扩增进行了平行实验。结果:2例胎儿均有SMNt基因第7、8外显子缺失;以羊水为模板和以基因组DNA为模板进行扩增的效率相似。结论:应用“HpHBuffer”直接对羊水样本中SMN基因上外显子7、8进行扩增,结合限制性片断长度多态性分析SMNt上是否发生缺失,可缩短诊断时间,节约诊断成本,减少检测过程中可能出现的样本交叉污染,有望成为临床上产前诊断脊肌萎缩症的新方法。

关 键 词:脊肌萎缩症  SMN基因  产前诊断  基因诊断  限制性片断长度多态性

Direct PCR from Amniotic Fluid for Rapid Diagnosis of Spinal Muscular Atrophy
HUANG Huan,WANG Jue,LU Shou-lian,HONG Lei,XIE Ai-ping,CAO Jun,SUN Li-zhou. Direct PCR from Amniotic Fluid for Rapid Diagnosis of Spinal Muscular Atrophy[J]. Biomagnetism, 2013, 0(35): 6845-6847,6903
Authors:HUANG Huan  WANG Jue  LU Shou-lian  HONG Lei  XIE Ai-ping  CAO Jun  SUN Li-zhou
Affiliation:(Department of Gynecology and Obstetrics, the First Atffliated Hospital o fNanjing Medical University, Nanjing, 210029, China)
Abstract:Objective: To establish a rapid and simple method to prenatally diagnose the spinal muscular atrophy (SMA). Methods: Based on the difference between two homologous copies(SMNc and SMNt) of survivat motor neuron gene(SMN), the deletion analysis ofexons 7 and 8 on gene SMNt was performed. Prenatal diagnosis of SMA was performed in 2 fetuses with SMA family history by using amniotic fluid directly without culturing and DNA extraction and a self-prepared "HpH-Buffer" with the method of PCR-Restriction fragment length polymorphism (RFLP). Furthermore, conventional PCR using genomic DNA extracted from each amniotic fluid sample was performed in parallel for evaluating the amplification efficiency of the experiments. Results: Deletion of exons 7 and 8 on gene SMNt was found in the two cases. The results of amplification efficiency from two kinds of starting material (amniotic fluid and extracted DNA) were consistent. Conclusion: Prenatal diagnosis of SMA using "HpH Buffer" to directly amplify the exons 7, 8 on gene SMNt and RFLP to analyze the deletion of SMNt could shorten the diagnosis time, save the diagnostic cost, avoid the cross-contamination during detection process. It was promising to become an efficient and rapid method of prenatal diagnosis for SMA.
Keywords:Spinal muscular atrophy  Survival motor neuron gene  Prenatal diagnosis  Genetic diagnosis  Restriction fragment length polymorphism
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