Facile solid-liquid biphasic PCR platform for ultrasensitive multiplex detection and genotyping of HPV

An innovative multiplex molecular detection method is introduced, ingeniously combining solid-phase PCR with liquid-phase PCR through the assistance of magnetic fluorescent-coded microspheres. In the amplification system, the liquid-phase PCR continuously generates target DNA fragments. Using these target DNA fragments as templates, specific primers on corresponding magnetic fluorescent-coded microspheres simultaneously perform solid-phase PCR. To comprehensively assess the performance of this multiplex nucleic acid detection method, we selected the quantitative detection of eight high-risk human papillomavirus (HPV) types (16, 18, 31, 33, 45, 56, 58, and 59) as a specific case for systematic investigation. This method demonstrated exceptional sensitivity, with a broad dynamic detection range from 6 × 10¹ to 6 × 10⁶ copies/µL with R² > 0.99. The limits of detection for this method were lower than 55 copies/µL, with the majority even below 40 copies/µL. Importantly, the developed method exhibited superior selectivity, wherein the amplified signal was detectable exclusively in the presence of the target nucleic acid. The consistency analysis revealed a strong correlation between the solid-liquid biphasic multiplex PCR assay and conventional qPCR, with correlation coefficients exceeding 0.99 in most cases. Additionally, detecting cervical exfoliated cell samples (without DNA extraction) yielded results consistent with hospital reports. In summary, the outstanding performance of the biphasic multiplex PCR assay makes it a promising technology for applications demanding multiple, ultrasensitive, and reliable detections, including clinical diagnostics, environmental monitoring, and food safety.