The sensitivity and specificity of allele-specific real-time PCR method used for NIPD.

Panel A shows one set of representative data from analysis of g.31921 T>C marker by allele-specific real-time PCR, in which the SNP profiling of artificial model samples is exported by amplification blot (left) and dissociation curve analysis (right). Our results showed superior sensitivity and specificity in all dilutions (see materials and methods), indicated by the arrows for each test, with a sensitivity of detection of 2 copies of the target sequence. Panel B shows the quantitative difference between the respective C_T values (ΔC_T) of maternal alleles (C_{T, maternal}) and paternal alleles (C_{T, paternal}) in analysis of g.31921T>C artificial model samples (see Materials and Methods). These results indicated a clear discrimination of the paternal allele from the maternal allele on experimental serial dilutions. Following the ΔC_{T,(paternal-maternal)} analysis, the maternal allele cluster is above a ΔCT value of 6, while the paternal allele cluster is at a value of less than 6, thus the arbitrarily assigned ΔCT cut-off value (gray dotted line) was used to distinguish between the presence of maternal from paternal alleles in the present study. Panel C shows the NIPD results obtained from detection of 65 at-risk fetus using 9 SNP markers in cfDNA as paternal-normal marker by allele-specific real-time PCR. Among 65 samples tested, correct appraisal of the presence (n = 33) or absence (n = 30) of the paternal-normal allele. Two cases labeled uncertain due to sample hemolysis by mishandling in the process of blood transport.