A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia
| Autor: | Fu Xiong, Shaoxiong Zhou, Ge Wang, Xiangmin Xu, Shun Xu, Xuefeng Zhao, Youming Cheng, Jianming Peng, Wanjun Zhou |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Genetics
Genotype Gene Dosage Reproducibility of Results Biology Real-Time Polymerase Chain Reaction Sensitivity and Specificity Molecular biology Gene dosage Pathology and Forensic Medicine Real-time polymerase chain reaction alpha-Globins alpha-Thalassemia Gene Order Multiplex polymerase chain reaction Gene cluster TaqMan Humans Mass Screening Molecular Medicine Multiplex Multiplex Polymerase Chain Reaction Genotyping Mass screening Multilocus Sequence Typing |
| Zdroj: | The Journal of Molecular Diagnostics. 15:642-651 |
| ISSN: | 1525-1578 |
| DOI: | 10.1016/j.jmoldx.2013.05.007 |
| Popis: | The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|