Department of Genetics, CHUS and Université de Sherbrooke, Sherbrooke, Québec, Canada.
Clin Biochem. 2012 Jan;45(1-2):88-91. doi: 10.1016/j.clinbiochem.2011.10.019. Epub 2011 Nov 7.
To describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood.
SMA is caused in most cases by homozygous deletion of the SMN1 gene. We describe a triplex quantitative real-time PCR method in which fragments of SMN1, SMN2 (a nearly-identical neighboring gene with markedly reduced function) and of a control gene, CFTR, are amplified in the same tube.
We validated this method in three ways. First, testing the same samples ten times yielded CV values <4.6%. Second, in 104 previously-genotyped individuals, SMN copy numbers identical to those of the previously-determined genotype was unambiguously obtained in all cases. Finally, results using the technique in practice are described and analyzed for reproducibility of amplification efficiency and for inter-run variability.
In over 1200 samples, this technique has proven accurate, fast, economical and reproducible.
描述并验证一种新的脊髓性肌萎缩症（SMA）分子诊断方案，这是一种常见的儿童神经肌肉疾病。
SMA 大多数情况下是由于 SMN1 基因纯合缺失引起的。我们描述了一种三重实时定量 PCR 方法，其中 SMN1、SMN2（一个功能显著降低的近同源邻近基因）和一个对照基因 CFTR 的片段在同一管中扩增。
我们通过三种方式验证了该方法。首先，对同一样本重复检测十次，CV 值均<4.6%。其次，在 104 名已进行基因分型的个体中，所有个体均明确获得与先前确定的基因型一致的 SMN 拷贝数。最后，描述了该技术在实际应用中的结果，并分析了扩增效率的重现性和运行间变异性。
在 1200 多个样本中，该技术已被证明准确、快速、经济且可重现。
