A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

• DOI: 10.1016/j.jmoldx.2011.03.005
• Publication Date: 2011-05-08T14:37:25Z
• Authors: 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
• Keywords: 0303 health sciences; DNA Mutational Analysis; beta-Thalassemia; Beta-Globulins; RAPID DETECTION; Reproducibility of Results; MASS-SPECTROMETRY; DNA; DIAGNOSIS; Polymerase Chain Reaction; CHINA; 3. Good health; 03 medical and health sciences; Double-Blind Method; Molecular Diagnostic Techniques; HEALTH BURDEN; Limit of Detection; 616; POPULATIONS; Humans; Transition Temperature; GLOBIN GENE-MUTATIONS; HEMOGLOBIN; POLYMORPHISMS

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 β-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 β-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 β-thalassemia mutations were accurately genotyped, and β-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 β-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.