Rapid and Sensitive Digital Droplet PCR Assays for Detecting HPV16 DNA in Liquid Biopsies

The combination of cell-free DNA (cfDNA) and digital droplet PCR (ddPCR) has significantly advanced the noninvasive screening, diagnosis, and monitoring of diseases, enabling highly sensitive and absolute quantification of target nucleic acids even in the presence of high background DNA. However, widespread adoption of ddPCR is hindered by higher costs, extended processing times, and the requirement for cfDNA purification, which adds expense and variability. To address these limitations, we developed two optimized ddPCR-based assays tailored for enhanced sensitivity, cost-efficiency, and ease of use. Our highly sensitive ddPCR assay for human papilloma virus (HPV)16 DNA detection in purified cfDNA from liquid biopsies from head and neck cancer patients significantly improved sensitivity by increasing cfDNA concentration by 8.5-fold, sample volume loading by 22-fold, and total cfDNA amount tested by 1200-fold without the need for restriction enzyme digestion. In parallel, we established a rapid ddPCR assay using unpurified cfDNA processed by heat treatment and centrifugation, achieving detection concordance rates of 55.6%, 66.7%, and 95.8% for plasma, serum, and surgical drain fluid (SDF), respectively, compared to purified cfDNA. Together, these complementary workflows, one optimized for unpurified cfDNA and the other for purified cfDNA, make ddPCR detection of specific targets in cfDNA more cost-effective, time-efficient, and standardizable across laboratories, paving the way for broader adoption in clinical diagnostics.