ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Biosensors and Biomolecular Electronics
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1608301
This article is part of the Research TopicBiomedical Sensing in Assistive DevicesView all 10 articles
Development of a One-Pot RPA-Cas12a/13a Assay for Simultaneous Detection of HPV16 and HPV18
Provisionally accepted
Qiujie Yu1,2
Hongzhi Luo3
Xiaoxue Huang2Xiaohua An1,2Yan Wang1,2Huasu Chen1,2Jianhuang Rong1,4Yafei Zhang1,2Qianhao Huang1,2Yudi Rao1,2
He Zha1,2*- 1Zunyi Medical University, Zunyi, China
- 2Department of Laboratory Medicine, The Third Affifiliated Hospital of Zunyi Medical University (The First People’s Hospital of Zunyi), Z, Zunyi, Guizhou Province, China
- 3Jintang First People's Hospital, Sichuan University, Jintang County, Sichuan Province, China
- 4Department of Oral Maxillofacial Surgery, School and Hospital of Stomatology, Zunyi Medical University,, Zunyi, Guizhou Province, China
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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/13abased fluorescence assays exhibited a sensitivity of 10 copies/µL, and required only 40 minutes 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: HPV, One-pot reaction, CRISPR-Cas12a/13a, Recombinase polymerase amplification, Multiplex detection
Received: 08 Apr 2025; Accepted: 03 Jul 2025.
Copyright: © 2025 Yu, Luo, Huang, An, Wang, Chen, Rong, Zhang, Huang, Rao and Zha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: He Zha, Zunyi Medical University, Zunyi, China
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