脊髓性肌萎缩症：一种用于测定 SMN1 和 SMN2 拷贝数的单管多重实时 PCR 检测方法的临床验证。
Spinal muscular atrophy: clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers.
机构信息
Department of Genetics, CHUS and Université de Sherbrooke, Sherbrooke, Québec, Canada.
出版信息
Clin Biochem. 2012 Jan;45(1-2):88-91. doi: 10.1016/j.clinbiochem.2011.10.019. Epub 2011 Nov 7.
OBJECTIVES
To describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood.
DESIGN AND METHODS
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.
RESULTS
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.
CONCLUSIONS
In over 1200 samples, this technique has proven accurate, fast, economical and reproducible.
目的
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。
设计与方法
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。
结果
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。
结论
在 1200 多个样本中，该技术已被证明准确、快速、经济且可重现。
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