Abstract
Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention.
Key points
• The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes.
• A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity.
• The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.
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Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
Code availability
Not applicable.
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Acknowledgements
We thank Huang Lab members for their helpful discussions. We are grateful for the technical support provided by the Core Facilities, Zhejiang University Medical Center/Liangzhu Laboratory, and the biotechnology platform of Zhejiang Laboratory.
Funding
This research was funded by the Key Research Project of Zhejiang Laboratory (2021PE0AC06), the National Natural Science Foundation of China (Grant No. 82002144, 81772533), and the Shanghai Municipal Science and Technology Commission (21N31900400).
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X.W., X.T., X.H., and B.H. conceived, designed, and supervised the project. B.H., Y.L., and Z.Z. performed most experiments with the help of X.K., Y.W., L.G., and M.W. X.S. provided expert technical assistance. B.H. and Y.L. wrote the paper with inputs from all authors. X.W., X.T., and X.H. revised the manuscript and managed the project.
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The study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (IIT2023014A). All research was performed under relevant guidelines and regulations. Informed consent was obtained from all participants.
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ESM 1
Figure S1: Sensitivity analysis of Cas12a-Based Fluorescent method of HPV11 with the original RPA primer volume. Figure S2: Clinical samples detection of sample #46 with the single tube test (with the qPCR Ct value of 36.71). Table S1: Information of the clinical samples tested in this study; Table S2: The DNA templates of six HPV subtypes for crRNA selection using the Cas12a-based fluorescence method; Table S3: The RPA primers of the six HPV subtypes used for screening. (PDF 355 kb)
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Huang, B., Lou, Y., Zeng, Z. et al. A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics. Appl Microbiol Biotechnol 107, 6287–6297 (2023). https://doi.org/10.1007/s00253-023-12728-5
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DOI: https://doi.org/10.1007/s00253-023-12728-5