Molecular diagnosis of α-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis

The aim of the study was to set up an automatic molecular diagnostic method for deletional α-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by dissociation curve (DC) analysis were used to detect the −−SEA, − α3.7, −α4.2, and non-deletion-type alleles (α α or αTα), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the dissociation curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon ≥ cutoff values. Molecular diagnosis of α-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the −−SEA, − α3.7, −α4.2, and non-deletion-type alleles were 82.5 ± 1°C, 82.8 ± 1°C, 81.5 ± 1°C, and 83.0 ± 1°C, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The CT VS log copies of a recombinant plasmid DNA showed a good linear relationship between 105 ∼ 100. Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 α-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of α-thalassemia.