A Nested Asymmetric PCR Melting Curve Assay for One-Step Genotyping of Nondeletional α-Thalassemia Mutations.

Abstract

A rapid DNA-based assay is essential for clinical diagnosis and mass screening in thalassemia-prevention programs. Because of high homology and guanine-cytosine-rich and complex second structure of α-globin genes, it is rather difficult to develop a feasible and simple method for α-thalassemia genotyping. In this study, a strategy of nested asymmetric PCR melting curve analysis was designed to tackle these factors and ensure sensitivity and accuracy. Herein, a novel one-step assay for genotyping of nondeletional α-thalassemia mutations, including hemoglobin (Hb) Westmead (HBA2: c.369C>G), Hb Quong Sze (HBA2: c.377T>C), Hb Constant Spring (HBA2: c.427T>C), CD30 (HBA2: c.91-93delGAG), and CD31 (HBA2: c.95G>A) in a single closed tube, was established and evaluated. All five mutations were accurately determined with the concordance rate of 100% in a blind analysis of 255 genotype-known samples and 1250 clinical samples. In conclusion, this assay is useful for rapid and reliable genotyping of nondeletional α-thalassemia mutations in clinical practice. Especially, the strategy may have the potential to be a versatile scheme for rapid genotyping of other gene mutations because of its high throughput, sufficient stability, low cost, and simple operation.

• Authors: Jiachun Qin, Mingli Xu, Qiang Zhang, Xiaojun Wen, Sheng He, Yong Zhou, Haiping Liu, Wanjun Zhou
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 22 Issue 6 Pg. 794-800 (06 2020) ISSN: 1943-7811 [Electronic] United States
• PMID: 32482310 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)
• Copyright: Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Chemical References

• Hemoglobins, Abnormal
• Ki-1 Antigen
• PECAM1 protein, human
• Platelet Endothelial Cell Adhesion Molecule-1
• alpha-Globins
• hemoglobin Quong Sze
• Hemoglobin Westmead
• Hemoglobin Constant Spring

Topics

• Data Accuracy
• Feasibility Studies
• Genotype
• Genotyping Techniques (methods)
• Hemoglobins, Abnormal (genetics)
• Humans
• Ki-1 Antigen (genetics)
• Mutation
• Platelet Endothelial Cell Adhesion Molecule-1 (genetics)
• Polymerase Chain Reaction (methods)
• Reproducibility of Results
• Sensitivity and Specificity
• alpha-Globins (genetics)
• alpha-Thalassemia (blood, genetics)