通过处理器介导的PCR提高高危型HPV基因型检测的灵敏度

High-risk human papillomavirus (hrHPV) genotypes, particularly HPV16 and HPV18, drive the majority of cervical and several anogenital and oropharyngeal cancers. Although molecular screening assays have grown more sensitive, major limitations remain. Current NGS-based methods offer genotype resolution but are costly, slow, and difficult to implement in routine screening. Digital PCR (dPCR) provides absolute quantification, but most assays target only one or two conserved regions, making it difficult to distinguish transforming infections from transient or non-oncogenic HPV. Additionally, HPV DNA in patient samples is often fragmented, integrated, or present at low abundance, complicating amplification and reducing both sensitivity and specificity. We developed a Processor-Mediated PCR that decouples amplification from fluorescent readout using a universal processor oligo and tunable universal probes. This enables high multiplexing regardless of instrument channel count. To improve specificity, we created iProbes, multi-domain probes that trigger fluorescent response only when their full target sequence is present, allowing discrimination between oncogenic and non-oncogenic HPV even within highly conserved regions. Using HPV16 as a model, we built a multi-fragment assay that detects 10 genomic regions (<90 bp each), including E6/E7 oncogenic segments, in a single dPCR or qPCR reaction. Processor-Mediated PCR is tunable to multiple applications and supports either multichannel or single-channel fluorescent readouts, ensuring compatibility with instruments with limited channel capacity. This broad molecular coverage enhances detection of fragmented or partially degraded viral genomes. Together, Processor-Mediated PCR and iProbe-based multiplexing provide sensitive, high-specificity detection of cancer-associated HPV fragments in one assay, addressing key limitations of current NGS and PCR methods and improving HPV screening and triage.