Dual-Strategy Optimization of Reference Systems Enhances Reliability in Tumor DNA Methylation Detection: Primer-Probe Innovation for Low-Concentration Clinical Specimens

ObjectiveThis study aimed to identify the optimal internal reference primer-probe set for multiplex qMSP, based primarily on amplification efficiency, to improve the detection pass rate for tumor DNA methylation analysis in low-DNA-concentration samples. MethodsOur screening strategy comprised three steps. First, we used SYBR Green-based qPCR to preselect primer pairs with robust amplification. Second, we applied probe-based qPCR to further evaluate these candidates and identify superior primer-probe combinations. Finally, we compared the amplification performance of the finalists across diverse sample types, including cell lines, tissues, blood cells, and plasma, to determine the optimal set. ResultsTwo optimal primer-probe sets were successfully identified: a performance-optimized set (bisACTB_103_F2+R2+P2) and a novel set (bisProAB1_F2+R2+P1). Both demonstrated superior amplification efficiency across various sample types. Crucially, the bisProAB1_F2+R2+P1 set significantly enhanced the amplification of low-concentration plasma cell-free DNA, thereby increasing the detection pass rate for minimal DNA input samples. ConclusionThis study provides two highly efficient internal reference systems for qMSP. The novel bisProAB1_F2+R2+P1 set, in particular, offers a substantial improvement for detecting low-concentration clinical samples, enabling better utilization of precious biospecimens.