Development of a Multiplex ddPCR Detection Method for Accurate Non-Invasive Prenatal Testing of Trisomy 21

Lijing Wang, Zunliang Wang, Xiaoqing Dai

Abstract

Down syndrome, induced by trisomy 21 (T21), manifests as cognitive impairment and significant fetal congenital anomalies. Noninvasive prenatal testing (NIPT) for trisomy 21 involves the identification of cell-free fetal DNA (cff-DNA) in maternal plasma. Current noninvasive prenatal testing (NIPT) technologies, primarily relying on next-generation sequencing (NGS), suffer from high costs and slow processing speeds. Recently, droplet digital PCR (ddPCR) stands out for its cost-effectiveness, high sensitivity, and rapid processing speed, emerging as a promising alternative to NGS for NIPT applications. But, the multiplex detection of cff-DNA from trisomy 21 using ddPCR often lacks sufficient specificity, mainly due to the complicated primers design. In this study, we present an optimized strategy for universal probe design through the screening of specific fragments at the whole-genome level. With this strategy, we designed 20 pairs of primers and a universal probe, specifically for targeting cff-DNA on chromosome 1 and 21. Our experimental results performed on simulation samples show that our developed ddPCR measurement method can effectively improve the detection specificity for cff-DNA from T21.