Novel real-time PCR assay for rapid prenatal diagnosis of Down syndrome: a prospective study of 563 amniocytes.

Objective: To explore the possibilities of a novel real-time PCR assay for rapid prenatal diagnosis of Down syndrome in clinical settings.

Design And Methods: This duplex real-time PCR assay is based on relative quantification of DSCR4 gene on chromosome 21 by using RABIF gene on chromosome 1 as a reference. For each sample, the differences in threshold cycles between DSCR4 and RABIF genes (Delta Ct, DeltaCt) were detected, and a calibrated DeltaCt value (DeltaDeltaCt, DeltaCt (sample)-DeltaCt (internal control)) was analyzed. Overall, 563 amniotic fluid samples from patients were blindly tested for fetal chromosome analysis and their DeltaDeltaCt values were evaluated according to the karyotyping results.

Results: Chromosome analysis revealed that 12 fetuses had trisomy 21 and 551 others were normal in chromosome 21. The DeltaDeltaCt values of trisomy 21 fetuses were significantly lower than those of normal ones (p-value<0.001) and no overlapping was shown: lower than -0.49 for trisomy 21 and above -0.30 for a normal one.

Conclusions: DeltaDeltaCt value could be used as a direct diagnostic index in real-time PCR assay; this novel assay is applicable for rapid prenatal diagnosis of Down syndrome in clinical settings.