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Background α-thalassemia is the most common inherited disorder of hemoglobin worldwide.A wide range of deletions occurred within human α-globin gene cluster represents the predominant determinant causing α-thalassemia.More rapid DNA assay directly applied on mass screening for deletional α-thalassemia is needed during thalassemia prevention program.We report a novel method that deletional α-thalassemia be rapidly genotyped with a quadruple qPCR gene dosage assay for α-globin gene copy number directly determination.Methods Three target genes(α2-,α 1-,ξ-globin)and a reference gene(CREBBP)amplicons on chroml 6p13.3 with very short size of 80-91bp were selected,subsequently a quadruple qPCR assay that contains four prime pairs and four-color TaqMan probes for target and reference genes was established,optimized and evaluated.678 clinical DNA samples containing 6 α-thalassemia deletions with 12 different α-globin genotypes and 186 normal samples,previously screened by MLPA or gap-PCR,were detected with this assay for accuracy and sensitivity evaluation.3000 random population samples of our local general population were furthermore detected with this assay and gap-PCR in double-blind analysis for deletional α-thalassemia population screening assessment.Results This assay had an equal amplification efficiency of each three target genes and reference gene,showed good accuracy with the mean intra-and inter-assay coefficient of variation 2.15-4.02 and 5.15-5.77respectively.Determined by 2-△△Cq method,the gene dosage ratios of 678 pretyped clinical DNA samples and 186 normal samples were 0.46-0.60 in the heterozygote,0.0 in the homozygote,0.97-1.07 in nondeleted genes for three target genes,respectively.It is shown that 100% concordance between this quantitative PCR and MLPA/gap-PCR method,including confirmation of all 12 different α-globin genotypes.The results of 3000 random local population screening showed the deletional α-thalassemia carrier rate is 9.6%,279 positive samples of different deletions and 2721 negative samples were fully coincident with the standard method,including identification of two novel deletions confirmed by MLPA.Conclusions This method is accurate,reproducible,and cost-effective in terms of equipment and reagents,assays using this method are simple to design and easy to perform,suitable for rapid genotyping and mass screening.