A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia
A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia
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DOI：
10.1016/j.jmoldx.2013.05.007
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Xu, Xiangmin
中科院分区：
文献类型：
--
作者：
Zhou, Wanjun;Wang, Ge;Xu, Xiangmin
The predominant determinants of alpha-thalassemia are deletions in the human alpha-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 alpha-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 alpha-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(-Delta Delta Cq) 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 alpha-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.
