A Stable Cell Line-Based Genomic DNA Reference Material for Accurate Hpv 45 Detection

Shujun Zhou, Hongbo Hu, Yuping Li, Wenjun Dai, Yi Yang, Jun Lin, Lianhua Dong

Abstract

The genomic DNA reference material (RM) for human papillomavirus (HPV) plays a pivotal role in enhancing the standardization, accuracy, and reliability of HPV DNA testing, which is essential for global cervical cancer screening and evaluating the efficacy of HPV vaccination programs. This study presents the development of a stable C-33A cell line integrating the full-length HPV 45 genome, establishing a robust foundation for our RM. An absolute quantitative method based on digital droplet PCR (dPCR) was developed for characterizing this RM, demonstrating high accuracy and analytical sensitivity with a limit of detection (LoD) of 8.57 and limit of quantification (LoQ) of 16.8 copies per reaction. The homogeneity and stability of the RM were confirmed over 24 months at −80 °C, with a certified value and expanded uncertainty (U, k = 2) of (1.56 ± 0.14) × 10³ copies/μL. Evaluation of inter-laboratory reproducibility across five platforms validated the consistency of HPV 45 copy concentrations, with a coefficient of variation (CV) of less than 5.0 %. This work establishes metrological traceability for HPV 45, enhancing quality control in testing laboratories and providing a reliable standard for HPV DNA detection assays. Importantly, the methods and findings of this study are not only applicable to HPV 45 but also provide a blueprint for developing reference materials for other high-risk HPV types, potentially significantly advancing HPV diagnostics and screening globally. In conclusion, our study significantly advances HPV diagnostics by developing a RM (NIM-RM 5256) and a dPCR method suitable for global standardized assessment of HPV DNA detection, potentially improving cervical cancer screening programs and the management of HPV-related diseases.