引物延伸变性高效液相色谱产前诊断β-地中海贫血

Accurate and rapid prenatal diagnosis of β-thalassemia by a multiplex primer extension and denaturing high performance liquid chromatography technique

目的建立针对中国人常见β-地中海贫血(β-地贫)基因型的产前诊断新方法。方法PCR扩增的靶序列,经引物延伸,得到中国人β-地贫5个常见突变的特异性延伸片段,用全变性高效液相色谱分析延伸片段混合物,分离图谱可鉴定被检样本的基因型。结果盲法分析显示,36个家系108例样品的引物延伸变性高效液相色谱与反向点杂交(reversedotblot,RDB)检测结果符合率为100%。其中6例RDB诊断为上述5个常见突变以外的突变。该法的突变检出率为94.4%(102/108),对产前诊断家庭的诊断率为97.2%(35/36)。结论该法是一种准确、高效的β-地贫突变分析方法,可用于β-地贫的产前诊断。

Objective To develop a primer extension in combination with denaturing high performance liquid chromatography (PE DHPLC) based assay for prenatal diagnosis of the five most common β thalassemia mutations in Chinese. Methods The human β globin gene fragment was amplified by PCR, followed by a multiple PE reaction specific for each five mutations. Then the PE product mixtures were separated for genotyping of β globin gene mutations using fully denaturing DHPLC analysis. Results In a blind study, prenatal diagnosis was performed on thirty six at risk families for β thalassemia major. Reverse dot blot (RDB) analysis was used to validate each result, showing an accuracy rate of 100% for PE DHPLC in a total of 108 samples tested. Overall, by PE DHPLC analysis, the authors could identify the genotypes involving the five mutations and normal alleles corresponding to 94 4% (102/108) and actually make final decision for prenatal diagnosis covering 97.2% (35/36). Conclusion The PE DHPLC protocol can be a simple, rapid, and highly accurate assay in the prenatal detection of common β thalassemia mutations.