Detection of three common mutations causing β-thalassemia by using a closed-tube multiplex PCR

Abstract

Small oligonucleotides mutations are the large majority causes of β-thalassemia. Dual priming oligonucleotide PCR has been used to detect point mutations and thus could be applied to diagnose β-thalassemia. The goal of this study was to establish a simple, quick and cost-effective screening assay by using modified dual priming oligonucleotide PCR for three most common mutations of β-thalassemia [CD71–72 (+A), CD 41–42 (-CTTT), Pnt.-28 (A → G)] in Southeast Asia and southern China. Man-made 5 tandem mismatched bases instead of poly (I) were used as the linker in the specific PCR primers. Single closed-tube multiplex PCRs followed by dissociation curve (DC) analysis were included in the molecular screening assay. Denaturing high-performance liquid chromatography analysis was applied to distinguish compound heterozygotes from single mutations. A blinded study of 91 samples was performed using this new assay. There were 41 samples detected as the above three mutations and it was concordant with the original methods. In conclusion, the modified dual priming oligonucleotide multiplex PCR/DC can detect these three genotypes of common mutation of β-thalassemia; this method is simple, rapid and cost-effective, which makes it suitable for large-scale screening and diagnosis.