A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics

作者：Huang, Baicheng; Lou, Yufeng; Zeng, Zihan; Kan, Xingchi; Shi, Xinping; Wu, Yue; Guo, Ling; Wang, Muzhen; Huang, Xingxu; Tian, Xuemei^*; Wang, Xinjie^*

来源：Applied Microbiology and Biotechnology, 2023, 107(20): 6287-6297.

DOI：10.1007/s00253-023-12728-5

• Human papillomavirus
• Diagnostic
• Recombinase polymerase amplification
• Cas12a
• Microfluidics

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.

• 单位
  浙江大学