人乳头瘤病毒（HPV）持续感染是子宫颈癌的主要原因，早期诊断对临床管理至关重要。然而，易于快速现场诊断HPV基因分型仍然具有挑战性。在这里，我们开发了一种基于Cas12a的荧光微流探测系统，用于诊断六种HPV亚型（HPV6，HPV11，HPV16，HPV18，HPV31和HPV33）。筛选了一组针对HPV L1基因的crRNA和重组酶聚合酶扩增（RPA）引物，以实现敏感和特异性检测。此外，开发了一种一锅RPA反应，可以放大六种HPV亚型而无交叉反应。为了现场检测，我们将RPA-Cas12a检测集成到微流控装置中，可以在35分钟内检测出处理过的临床样本。该检测方法使用了112个临床拭子样本进行验证，并与qPCR检测方法得到一致的结果，一致性率为99.1%。总体而言，我们的诊断方法为检测HPV基因型提供了一种快速、敏感和易于使用的现场检测方法，并有望改善子宫颈癌筛查和预防。关键点：•基于Cas12a的荧光微流探测系统可用于诊断六种HPV亚型。•一锅式RPA反应可放大六种HPV亚型而无交叉反应。•RPA-Cas12a-微流控系统可在35分钟内获得现场检测结果。© 2023 Springer Nature独家许可给Springer-Verlag GmbH Germany。

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.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.