Multiplex Visualized Closed-Tube PCR with Hamming Distance 2 Code for 15 HPV Subtype Typing

Cervical cancer is the fourth leading cause of death in women, especially in developing countries. Specific and economic methodologies for HPV typing are crucial in cancer diagnosis and further disease control. However, routine assays based on real-time polymerase chain reaction (qPCR) or DNA-chip hybridization are either incapable of offering detailed subtype information or involve tedious open-tube operations with the risk of cross-contamination from PCR amplicons. Herein, we proposed a multiplex visualized closed-tube PCR (Multi-Vision) for HPV typing. Using gold nanoparticle probes (AuNPs) as a color change indicator combined with a Hamming distance 2 coding scheme, 13 high-risk HPVs and two subtypes associated with high-incidence benign lesions were successfully typed by performing six closed-tube PCRs. The assay demonstrates high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 10⁰ to 10³ copies per reaction) and enables highly sensitive detection of as low as 0.5 copies/μL. Further, 105 clinical samples were successfully analyzed using our method with a high concordance rate of 99.05% (104/105) compared to a HPV typing kit. The inconsistent sample was confirmed by sequencing to be consistent with the typing results determined by our method, indicating that Multi-Vision could be a useful tool for HPV detection, especially in resource-limited regions.