High-throughput β-thalassemia carrier screening by allele-specific Q-primer real-time polymerase chain reaction

Abstract

Based on a novel Q-primer real-time polymerase chain reaction (PCR) system, we designed allele-specific Q-primers for the detection of three β-thalassemia mutations [Cd41/42(-TCTT), IVSI nt5 (G>C), and IVSII nt654 (C>T)] that have a high carrier frequency in Southeast Asia. With clear distinction between heterozygote and wild-type, Δ C t (threshold cycle) values were defined. The results of evaluating 139 blinded samples by our system match perfectly with those obtained by the conventional reverse dot blot (RDB) method. With a 384-well plate that included replicates in the same analysis, our throughput reached 190 reactions per run with a turnaround time as short as 130 min, and the cost of consumables was as low as $1 (US) for each test.