Dual-Strategy qMSP Reference Optimization for Low-DNA Clinical Specimens.

Detecting tumour DNA methylation in low DNA concentration samples at high qualification rates is a significant challenge in translating and conducting regulatory trials for tumour methylation detection kits. Existing internal reference systems frequently malfunction, resulting in the mislabelling of valuable clinical specimens as unusable. We developed a two-part framework for improving reference primer-probe sets, by combining a systematic improvement of existing ACTB assays with new primer designs that target previously unexplored, CpG-free genomic areas. A three-stage screening process involving SYBR Green qPCR, TaqMan probe-based qPCR, and validation across different biological samples led to the identification of two superior primer sets: an optimised combination (bisACTB_103_F2+R2+P2) and a novel set (bisProAB1_F2+R2+P1). Exhibited enhanced amplification efficiency and specificity in cell lines, tissues, blood cells, and plasma were observed in both. Notably, bisProAB1_F2+R2+P1 considerably enhanced amplification efficiency in low-concentration plasma cell-free DNA, resulting in a nearly 5% increase in the detection success rate within a large clinical study group and allowing for reliable analysis of minimal-input samples. This work offers verified, high-performance reference systems to boost the reliability of DNA methylation-based diagnostics and make the most of precious biospecimens.