Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

摘要

Background: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods.Methods: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene.Results: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY . For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%–22% when amplifying DYS14 compared with 26%–140% for SRY .Conclusions: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.