A Melting Curve Analysis-Based PCR Assay for One-Step Genotyping of B-Thalassemia Mutations:: A Multicenter Validation

• Autores: Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua Zhang, Ren Cai, Yajun Chen, Jiansheng Xie, Shanwei Feng, Xiaofeng Wei, Qizhi Xiao, Tianlang Zhang, Shiqiang Luo, Xuehuang Yang, Ying Hao, Yanxia Qu, Qingge Li, Xiangmin Xu
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 13, Nº. 4, 2011, págs. 427-435
• Idioma: inglés
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• Resumen

  • The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 B-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of B-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 B-thalassemia mutations were accurately genotyped, and B-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple B-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost.