Electrochemical LAMP platform for detecting HPV integration at the mRNA level in cervical cancer

Background: Human papillomavirus (HPV) is a double-stranded DNA virus linked to epithelial tissue infections and various cancers, notably cervical cancer. The integration of high-risk HPV types like HPV16 into the host genome is a critical factor in cancer development. Traditional methods for detecting HPV integration, such as immunohistochemistry (IHC) and PCR-based assays, are complex and time-consuming. Thus, alternative tools are being developed to speed up and simplify the analysis. Our study introduces an innovative RNA-biosensing platform that combines loop-mediated amplification (LAMP) with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript, which is lost upon integration. 

Materials and methods: Total RNA was extracted and amplified using single-step RT-LAMP to produce DNA products labeled with digoxigenin. These were mixed with streptavidin magnetic beads coated with biotinylated capture probes. After hybridization, anti-digoxigenin antibodies conjugated to HRP enabled the amperometric readout. Using an eight-electrode array, we significantly reduced the overall time by simultaneously analyzing eight samples. A magnet beneath the array concentrated the HRP molecules by attracting the magnetic beads to the electrode surface. Results: Our platform demonstrated distinct expression patterns of E7 and E2 mRNAs in HPV16-positive cell lines. EC readouts showed high E7 expression in HPV16-positive cell lines and a loss of E2 expression in fully integrated HPV genomes. Clinical samples confi rmed the ability of the platform to diff erentiate between integrated and episomal forms of HPV16. Comparison with standard techniques, including ddPCR and IHC, showed high correlation, validating the accuracy and reliability of our method.

Discussion: The eRT-LAMP method provides a simple, rapid, and cost-effective alternative for determining HPV integration status, crucial for assessing the risk of disease progression in women with precancerous cervical lesions. The integration of LAMP with EC detection offers a significant reduction in analysis time compared to traditional methods, making it suitable for point-of-care diagnostics and personalized medicine.

We acknowledge support of the Czech Health Research Council (No. NU21-08-00057), National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) – Funded by the European Union – Next Generation EU.