A melting curve analysis--based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation

J Mol Diagn. 2011 Jul;13(4):427-35. doi: 10.1016/j.jmoldx.2011.03.005. Epub 2011 May 6.

Abstract

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.

Publication types

  • Multicenter Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Beta-Globulins / genetics*
  • DNA Mutational Analysis / methods*
  • Double-Blind Method
  • Humans
  • Limit of Detection
  • Molecular Diagnostic Techniques*
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Transition Temperature*
  • beta-Thalassemia / diagnosis*
  • beta-Thalassemia / genetics

Substances

  • Beta-Globulins