Development of a novel MSRE-dPCR assay for non-invasive prenatal testing of trisomy 21

Currently, next-generation sequencing (NGS)-based non-invasive prenatal diagnosis testing (NIPT) of fetal T21 has been proven to be much more advantageous than traditional serum biochemical tests in terms of accuracy and sensitivity. Nevertheless, serum biochemical tests remain the first choice for the screening of trisomy 21 (T21) since NGS is too expensive and time-consuming. In this respect, this paper proposes a methylation-sensitive restriction endonuclease (MSRE)-digital PCR (dPCR) method for the screening of T21, which enriches the fraction of fetal-specific DNA by digesting maternal sequences and could more easily reflect the fold changes of chromosome 21. For this purpose, 64 DMRs on chromosome 21 and the control chromosome were tested for their ability to detect T21 with MSRE-dPCR. After MSRE digestion, 8 chromosome 21-specific DMRs and a chromosome 1 reference DMR (CD48) exhibited significant differences between fetal and maternal DNA, which were then applied for multi-index detection <i>via</i> dPCR. After testing 24 simulated samples, the corresponding calculation formulas were established for MSRE-dPCR (a 4-marker panel), and the distinguishing accuracy was 100%, which was much better than that of MSRE-dPCR (87.5%). Finally, the detection limit of MSRE-dPCR was found to be 2.44-4.76%. In a preliminary validation using clinical cffDNA samples, the established method correctly classified 18 out of 19 cases (94.73% accuracy), distinguishing T21 from healthy pregnancies. This MSRE-dPCR strategy provides a rapid, cost-effective alternative with promising accuracy for T21 detection, serving as a potential supplementary tool in prenatal screening. Further validation with larger cohorts is warranted to confirm its clinical utility.