Design and development of a diagnostic method for HPV-16 virus with nanoball DNA based on vertical flow assay.

The Human papillomavirus is one of the deadliest causes of cancer in women. This study aimed to design and develop a diagnostic method based on Rolling Circle Amplification (RCA) and vertical Flow Assay (VFA) for detection of HPV16. DNA nanoball synthesis was optimized using Phi29 DNA polymerase and probes. Nitrocellulose membranes were saturated with saline-sodium citrate buffer (SSC), and different probe concentrations were tested for optimal color production and intensity measurement. The results were checked with Image J software. Membrane blocking and washing buffer formulations were optimized to minimize background noise and increase test accuracy. Real-time PCR was used as a gold standard, confirming 39 HPV16-positive clinical samples. The assay was validated with positive HPV16 and HSV samples, and the sensitivity and specificity of the VFA-RCA method were determined. The limit of detection (LOD) for HPV16 was 0.5 ng, equivalent to 472 DNA copies/ml, with a sensitivity of 94 % and a specificity of 88 %. The RCA-VFA method offers a reliable, sensitive, and specific diagnostic approach for the early detection of HPV16, demonstrating potential to identify cancer at an early stage.