A melting curve analysis--based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation
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...
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| Veröffentlicht in: | The Journal of molecular diagnostics : JMD 2011-07, Vol.13 (4), p.427-435 |
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| creator | Xiong, Fu Huang, Qiuying Chen, Xiaoyun Zhou, Yuqiu Zhang, Xinhua Cai, Ren Chen, Yajun Xie, Jiansheng Feng, Shanwei Wei, Xiaofeng Xiao, Qizhi Zhang, Tianlang Luo, Shiqiang Yang, Xuehuang Hao, Ying Qu, Yanxia Li, Qingge Xu, Xiangmin |
| description | 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. |
| doi_str_mv | 10.1016/j.jmoldx.2011.03.005 |
| format | Article |
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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.</description><identifier>ISSN: 1525-1578</identifier><identifier>EISSN: 1943-7811</identifier><identifier>DOI: 10.1016/j.jmoldx.2011.03.005</identifier><identifier>PMID: 21704277</identifier><language>eng</language><publisher>United States: American Society for Investigative Pathology</publisher><subject>Beta-Globulins - genetics ; beta-Thalassemia - diagnosis ; beta-Thalassemia - genetics ; DNA Mutational Analysis - methods ; Double-Blind Method ; Humans ; Limit of Detection ; Molecular Diagnostic Techniques ; Polymerase Chain Reaction ; Regular ; Reproducibility of Results ; Transition Temperature</subject><ispartof>The Journal of molecular diagnostics : JMD, 2011-07, Vol.13 (4), p.427-435</ispartof><rights>Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.</rights><rights>2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. 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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. 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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. 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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Access via ScienceDirect (Elsevier); Alma/SFX Local Collection |
| subjects | Beta-Globulins - genetics beta-Thalassemia - diagnosis beta-Thalassemia - genetics DNA Mutational Analysis - methods Double-Blind Method Humans Limit of Detection Molecular Diagnostic Techniques Polymerase Chain Reaction Regular Reproducibility of Results Transition Temperature |
| title | A melting curve analysis--based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation |
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