Analysis of Real-Time SYBR-Polymerase Chain Reaction Cycle Threshold for Diagnosis of the α-Thalassemia-1 Southeast Asian Type Deletion: Application to Carrier Screening and Prenatal Diagnosis of Hb Bart's Hydrops Fetalis

Abstract

Without gel electrophoresis and specific probes, the two tubes real-time SYBR-polymerase chain reaction (SYBR-PCR) was setup by using different primer sets: P1/P2 for the detection of wild type α-globin gene alleles and P1/P3 for detection of the allele bearing the Southeast Asian (SEA) type (– –^SEA) deletion. Analyses of the cycle threshold (C_T) values obtained by each primer set together with a delta-cycle threshold (ΔC_T) and C_T ratio, showed that lower C_T values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Bart's hydrops fetalis subjects, respectively. In heterozygous subjects the C_T values generated by both sets of primers were similar to each other. There was no overlapping of ΔC_T and C_T ratio between normal, heterozygous and Hb Bart's hydrops fetalis subjects. Therefore, the two tubes real-time SYBR-PCR could represent a rapid, cost effective, high-throughput assay for screening of carriers and prenatal diagnosis of α-thalassemia-1 (α-thal-1) with the SEA type (– –^SEA) deletion.

Keywords:

• Real-time SYBR-polymerase chain reaction (SYBR-PCR)
• Cycle threshold (C_T)
• α-Thalassemia-1 (α-thal-1)
• SEA type deletion
• Hb Bart's hydrops fetalis