PVA sponge method improves detection efficiency of cfDNA in plasma

Cell-free DNA (cfDNA) analysis is an important tool in molecular diagnostics and translational research. However, conventional cfDNA extraction from plasma is often labor-intensive, and inefficient due to interference from blood-derived substances. We developed a novel cfDNA sample preparation method using polyvinyl alcohol (PVA) sponges that enables direct qPCR without extraction or purification.

PVA sponge pieces were prepared and used to dry plasma or DNA standard solutions. After drying, the sponge was directly added to the qPCR mixture. Recovery yields were evaluated using human genomic DNA and 200 bp synthetic DNA at various concentrations and compared with two commercial extraction kits: QIAamp DNA Blood Mini (DBM) and QIAamp MinElute ccfDNA (MEC). Clinical plasma samples from 10 patients were also analyzed.

PVA sponges did not interfere with qPCR amplification, achieving recovery yields of 90.7% - 105.3% for genomic DNA. In plasma-spiked samples, qPCR inhibition was mitigated by using PVA sponges, resulting in improved amplification curves and reduced Ct variability. For low-concentration synthetic DNA (10¹ copies/μL), recovery yields were 46.0% with the PVA sponge method, compared to 11.4% and 30.9% with DBM and MEC, respectively. The PVA sponge method required only four steps and about one hour, significantly fewer steps than commercial kits. Median cfDNA concentration in patient plasma was 7.8 copies/μL.

The PVA sponge method enables efficient and rapid cfDNA quantification directly from plasma without extraction. Its simplicity, speed, and cost-effectiveness make it a promising tool for liquid biopsy applications in clinical practice.