Special RCA based sensitive point‐of‐care detection of HPV mRNA for cervical cancer screening

Abstract

Developing the sensitive point‐of‐care testing (POCT) of oncogenic nucleic acids from human papillomavirus (HPV) infection is essential in preventing cervical cancer, especially in resource‐limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT via nucleic acid‐based aggregation under isothermal conditions. However, the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity. Here, a minimum secondary structured RCA technique (MSS‐RCA) is developed by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions. The amplification efficiency of MSS‐RCA could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS‐RCA, 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 nucleic acid biomarkers for screening of cervical cancer or other diseases.

Portable Device with Nicking Enzyme Enhanced Special RCA on μPADs toward Sensitive Detection of High-Risk HPV Infection

“…However, the specific design of the circular template employed in MSSRCA restricts its applicability in target detection. Additionally, due to its linear amplification format, MSSRCA exhibits inferior sensitivity when compared to the exponential and cascade RCA-based methodologies mentioned above . The integration of a nicking enzyme with RCA has resulted in the development of a highly efficient amplification strategy known as nicking-enhanced RCA (N-RCA).…”

“…Additionally, due to its linear amplification format, MSSRCA exhibits inferior sensitivity when compared to the exponential and cascade RCA-based methodologies mentioned above. 24 The integration of a nicking enzyme with RCA has resulted in the development of a highly efficient amplification strategy known as nickingenhanced RCA (N-RCA). While N-RCA retains the advantages of simplicity, previous approaches have certain limitations that hinder their suitability for POC scenarios, including prolonged reaction times and limited portability due to the necessity of a heating block and signal readout.…”