Application of real‐time PCR–based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses

To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program.A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated.Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay.MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.