A multiplex digital PCR-based all-in-one non-invasive prenatal test for simultaneous detection of Fetal chromosomal aneuploidies and Fetal fraction quantification

Current approaches for prenatal aneuploidy detection include conventional serum-based biochemical assays and non-invasive prenatal testing (NIPT) based on next-generation sequencing (NGS). Serum-based assays, though economically feasible and rapid, demonstrate insufficient accuracy and extended processing durations (e.g., karyotyping requiring up to 2 weeks). While NIPT offers high sensitivity for detecting common aneuploidies (e.g., T21/T18/T13) with 100 % specificity, its clinical implementation is hindered by high costs, technical complexity (e.g., requiring specialized library preparation and bioinformatics analysis), and restricted accessibility in resource-limited settings. In this study, we developed a multiplex digital PCR NIPT (dPCR-NIPT) approach based on an all-in-one digital PCR platform that automates partitioning, PCR amplification, and fluorescence detection, enabling simultaneous detection of fetal T21, T18, or T13 and quantification of cell free fetal DNA (cffDNA) within a single tube. The assay demonstrated exceptional analytical performance with detection limits of 5 % cffDNA and minimal input requirements of 5 ng cfDNA. Compared with NGS-NIPT, our dPCR-NIPT detected 180 plasma samples from pregnant women, with a sensitivity and specificity of 100 % and 100 %, respectively, and an average fetal fraction of 15.14 %. Notably, this performance was maintained across varying DNA concentrations and gestational stages. The automated workflow reduces manual intervention and potential contamination risks, while the low input requirement (5 ng cfDNA) enhances its applicability in clinical settings with limited sample availability. The novel multiplexing capability of the all-in-one dPCR-NIPT system enables simultaneous detection of fetal T21/18/13 and cffDNA quantification within a single tube, offering superior analytical performance and operational simplicity compared to existing NGS-based methods. This integration addresses critical limitations of traditional serum assays (low accuracy) and NGS-NIPT (high cost, technical complexity), while maintaining 100 % sensitivity/specificity in clinical validation. These characteristics position it as a promising candidate for routine prenatal screening applications. A fully automated multiplex ddPCR system enables simultaneous detection of T21/T18/T13 trisomies and fetal fraction quantification. This integrated platform combines methylation-dependent cffDNA enrichment and multi-color fluorescence detection, streamlining prenatal screening with ultra-high analytical precision (5 % fetal fraction limit) and rapid turnaround time (4 h). • An automated multiplex digital PCR system was established to detect T21/T18/T13 and quantify fetal fraction. • A 60-fold multiplex ddPCR system achieves high accuracy for T21/T18/T13 detection with 5 ng of maternal plasma input. • The system achieves a detection limit of 5 % fetal fraction with 5 ng cfDNA input. • Validation with 180 maternal plasma samples demonstrated 100 % sensitivity/specificity vs. NGS-NIPT, with results in < 4 h. • This system eliminates manual partitioning and multi-step procedures, reducing complexity and improving clinical workflow.