Development of a Nano-Real-Time Polymerase Chain Reaction (RT-PCR) Kit for Detection and Genotyping of High-Risk Human Papillomavirus (HPV) Strains Using Dedicated TaqMan Probes

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BACKGROUND: Gold nanoparticles (AuNPs) have demonstrated promise in enhancing polymerase chain reaction (PCR) efficiency, leading to more precise viral detection. The integration of AuNPs into PCR protocols has been shown to improve amplification efficiency and detection specificity, thereby enabling more accurate viral diagnostics. This study developed and optimized a nano-real-time PCR (RT-PCR) assay incorporating AuNPs for human papillomavirus (HPV) detection and genotyping. METHODS: The synthesized pegylated AuNPs were characterized using ultraviolet-visible spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential analysis, and Fourier-transform infrared spectroscopy (FTIR). Different concentrations of AuNPs were tested to determine the optimal conditions of RT-PCR. The specificity and sensitivity of the assay were evaluated by testing various HPV genotypes and comparing the performance with commercial PCR kits. RESULTS: The results confirmed that the optimal concentration of AuNPs for improved PCR performance was 1 nM, ensuring stable amplification efficiency across different fluorophore-labeled TaqMan probes (FAM, HEX, ROX, and Cy5). The assay successfully detected HPV DNA at concentrations as low as 0.1 ng and demonstrated 100% specificity, with no cross-reactivity observed with herpes simplex virus (HSV). The detection limit of the assay was 16 copies per reaction, surpassing conventional PCR methods. Additionally, the designed kit showed comparable accuracy to commercial detection kits, further supporting its diagnostic reliability. CONCLUSION: Incorporating AuNPs in RT-PCR significantly improved fluorescence signal detection, primer binding efficiency, and polymerase activity, enhancing sensitivity and specificity. Given its high diagnostic accuracy, this assay represents a promising tool for the early and reliable detection of HPV, with potential applications in clinical diagnostics and epidemiological surveillance.

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