Selenium-enhanced recombinase polymerase amplification-CRISPR/Cas12a: A low-noise single-tube assay for human...

In vitro amplification represents a critical step in human papillomavirus (HPV) DNA detection. However, DNA polymerases can initiate nonspecific amplification and incorporate erroneous nucleotides due to the lack of cellular repair mechanisms. To address these challenges, we present a novel one-tube selenium-enhanced recombinase polymerase amplification (Se-RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 12a (Cas12a) (OTSRC) system for the ultrasensitive HPV DNA detection. The Se-RPA incorporates 10 % selenium-modified nucleoside triphosphates (dNTPαSe) into the conventional RPA protocol, effectively suppressing nonspecific amplification while maintaining high-fidelity DNA synthesis. The CRISPR/Cas12a component integrates sequence-specific verification, exponential signal amplification, and fluorescence-based readout capabilities. Optimized in a single-tube format to minimize aerosol contamination, OTSRC exhibits a background signal of 71.77 % compared to the one-tube RPA-CRISPR/Cas12a (OTRC) system. Within a 20-min incubation, the OTSRC demonstrated a detection limit of 169 aM, which is half that of the OTRC without dNTPαSe and comparable to qPCR. Furthermore, the OTSRC system demonstrates the excellent compatibility of dNTPαSe with the RPA-CRISPR/Cas12a system, thereby enhancing HPV detection sensitivity. Overall, OTSRC enables rapid, sensitive, and specific detection of HPV DNA, showing strong potential for clinical point-of-care nucleic acid testing applications.