UPCRISPR: an ultrafast, one-tube multiplexed nucleic acid detection system for resolving the diagnostic gray zone in HPV16/18 detection.

Detection of high-risk human papillomavirus (HPV) is crucial for early cervical cancer screening. Polymerase chain reaction (PCR) is widely regarded as the gold standard for nucleic acid detection. However, there are still several constraints, such as long turnaround time and the "gray zone" issue. In this study, an ultrafast one-tube multiplex detection method called UPCRISPR was developed for highly sensitive simultaneous detection of HPV16 and HPV18. The entire detection process was completed in ∼10 min. By integrating ultrafast multiplex PCR and Cas12a cleavage, it simultaneously detected as few as 10 copies/μL of HPV16 and 100 copies/μL of HPV18. The proposed method was further validated using 130 clinical samples for practical applications. In 100 routine clinical samples, the UPCRISPR results showed 100% agreement with a commercial quantitative PCR kit used as the comparison method. In addition, all 30 qPCR gray-zone samples were correctly classified relative to qPCR combined with melting curve analysis. In summary, the proposed UPCRISPR method exhibits excellent sensitivity, specificity, speed and ease of use, demonstrating great potential for widespread application in cervical cancer screening.