Development of a one-pot RPA-cas12a/13a assay for simultaneous detection of HPV16 and HPV18

Abstract

The incidence of human papillomavirus (HPV)-related cervical cancers has been on the rise, and the affected population is increasingly younger. Early-stage prevention and screening initiatives have emphasized the critical necessity for reliable and rapid HPV detection technique. In this study, we devised a fluorescence-based assay that integrated one-pot Cas12a/13a with recombinase polymerase amplification (RPA) for the detection of HPV16 and HPV18. We exploited the cleavage activities of the Cas12a and Cas13a enzymes to specifically target the L1 gene of HPV16 and 18, respectively. The diagnostic efficacy of the CRISPR-Cas12a/13a system was assessed in identifying HPV by analyzing clinical samples and comparing it with the PCR method. The one-pot RPA-Cas12a/13a-based fluorescence assays exhibited a sensitivity of 10 copies/µL, and required only 40 min for completion. Compared with PCR method, the overall sensitivity and specificity of this assay were 97.69% and 100%, respectively, with a kappa value of 0.967. This study presents a novel approach for cervical cancer screening and HPV infection surveillance, which may hold potential for the early diagnosis and prevention of HPV-related cervical malignancies.

Keywords: CRISPR-Cas12a/13a; HPV; multiplex detection; one-pot reaction; recombinase polymerase amplification.

FIGURE 1

Schematic diagram of the one-pot RPA-Cas12a/13a assay for the simultaneous detection of HPV16 and HPV18. The CRISPR system was placed at the cap of the tube, while the RPA components were added to the bottom for amplification at 37°C for 20 min. After brief centrifugation, the CRISPR system was transiently dissociated to the bottom of the tube to react with the amplification products for 20 min, enabling readout of real-time fluorescence signals in optical instruments. Created in BioRender. Qiujie, Y. (2025) https://BioRender.com/m60g599.

FIGURE 2

Optimization of the RPA reaction system. (A) The RPA reaction volume was optimized from 10, 15, 20, 25, and 50 μL. (B) The RPA reaction time was optimized from 10, 15, 20, 25 and 30 min. NC indicates nuclease-free water.

FIGURE 3

Orthogonal experiments with the CRISPR system. (A) Both ssDNA reporter (FAM-BHQ) and ssRNA reporter (ROX-BHQ) were added to the detection systems of Cas12a, and changes in the fluorescence intensity of both types were observed. The activated Cas12a cleaved only the ssDNA reporter, generating a FAM fluorescent signal. (B) Both ssDNA reporter (FAM-BHQ) and ssRNA reporter (ROX-BHQ) were added to the detection systems of Cas13a, and changes in the fluorescence intensity of both types were observed. The activated Cas13a cleaved only the ssRNA reporter, generating a ROX fluorescent signal. (C) The schematic diagram of the dual-gene detection system for HPV16/18 with LbaCas12a and LwaCas13a. NC indicates nuclease-free water (****P < 0.0001). Created in BioRender. Qiujie, Y. (2025) https://BioRender.com/m60g599.

FIGURE 4

Optimization of the CRISPR system. (A) Optimization of Cas12a to crRNA concentration ratio for the Cas12a system in HPV16 L1 genetic testing. (B) Optimization of the reaction time of CRISPR/Cas12a from 10, 15, 20, 25 and 30 min. (C) Optimization of Cas13a to crRNA concentration ratio for the Cas13a system in HPV18 L1 genetic testing. (D) Optimization of the reaction time of CRISPR/Cas13a from 10, 15, 20, 25 and 30 min. (E) Optimization of rNTPMix solution concentration in the Cas13a cleavage system. (F) Optimization of T7 RNA polymerase concentration in the Cas13a cleavage system. (G) Optimization of RPA and CRISPR reaction temperatures from 35, 37, 39, 42, 45°C. NC indicates nuclease-free water (ns > 0.05, *P < 0.05, *P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 5

Sensitivity and specificity analysis of one-pot RPA-Cas12a/13a assay. (A) Sensitivity of the one-pot Cas12a cutting system for detection of the HPV16. (B) Sensitivity of the one-pot Cas13a cutting system for the detection of HPV18. (C) Sensitivity of the one-pot RPA-Cas12a/13a dual system for detection of HPV16/18.

FIGURE 6

Specificity assessment of the one-pot RPA-Cas12a/13a assay. The specificity was tested with DNA samples from HPV 16, 18, 33, 52, 39, 51, 58 and RNA samples from HCV.

FIGURE 7

Evaluation of the one-pot RPA-Cas12a/13a assay on HPV samples. (A) Scatter plot of the normalized PL intensity of 150 clinical samples tested with the one-pot RPA-Cas12a/13a assay. HPV16-positive samples are indicated by blue dots, and HPV16-negative samples are indicated by red dots. (B) Scatter plot of the normalized PL intensity of 150 clinical samples tested with the one-pot RPA-Cas12a/13a assay. HPV18-positive samples are indicated by blue dots, and HPV18-negative samples are indicated by red dots.