羊水直接PCR法快速诊断脊肌萎缩症的实验研究

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。

Direct PCR from Amniotic Fluid for Rapid Diagnosis of Spinal Muscular Atrophy

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.