CRISPR-Cas12a Coupled with Universal Dual-mode Fluorescent Nanoparticles Platform for HPV Nucleic Acid Detection

Cervical cancer is a prevalent gynecological malignancy, with approximately 90% of cases attributed to human papillomavirus (HPV) infection. Rapid and accurate nucleic acid detection is one of the leading methods to improving screening coverage for early cervical cancer diagnosis. However, most existing techniques are usually complex and require expensive instrumentation. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems have great advantages in nucleic acid detection. We herein combined the CRISPR-Cas12a with a universal dual-mode fluorescent nanoparticles (FNPs) platform to construct a highly sensitive signal-off assay for HPV high-risk subtypes detection. The signal readout module uses a single-stranded DNA linker, which forms a sandwich structure with DNA-functionalized magnetic beads and DNA-functionalized FNPs to generate signals. If trans-cleavage activity was activated by the targets, the linker was consumed and therefore could not form the sandwich structure to produce signals. HPV16 and HPV18 as model targets, the limits of detection as low as 5 pmol/L were successfully achieved without amplification. We validated the feasibility of the real-sample detection using HPV16 and HPV18 pseudo viruses. The proposed method can be easily adapted for other virus or bacterial assays by modifying the CRISPR-derived RNA (crRNA), which shows great potential for clinical diagnosis.