A Droplet Digital PCR Assay Targeting Sixteen Human Papillomavirus Genotypes

Background Cervical screening with high precision assays such as HPV DNA testing is essential for detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes. Methods Primers and probes were designed to target the E6 region of 16 HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards, and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit (IU) or genome equivalent (GE) per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7/234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.