MSSRCA Powered Sensitive Point-of-Care Detection of HPV mRNA for Cervical Cancer Screening

Developing sensitive point-of-care testing (POCT) of oncogenic nucleic acids from HPV infection is essential in preventing cervical cancer, especially in resource-limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT; however, still exists a bottleneck requiring multiple amplification steps with multiple detection probes due to the limited sensitivity. Here we developed a minimum secondary structured RCA technique (MSS-RCA) by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer. We demonstrated that RCA amplification efficiency could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS-RCA, Human Papillomavirus 16 (HPV16) E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fM (50 zmol in 10 µL). This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS-RCA-PGM provides a sensitive POCT platform for the detection of biomarkers for screening of cervical cancer or other diseases.