Development of Rapid Detection Technology for HPV16 Based on CRISPR-Cas12a

Abstract

Objective: Cervical cancer is one of the most common malignant tumors affecting women. It is estimated that 99.6% of cervical cancer patients are caused by persistent infection with human papillomavirus (HPV). The aim of this study was to combine the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated proteins (Cas12a) detection system with colloidal gold rapid diagnosis technology to develop a new rapid diagnosis technology for HPV16 nucleic acid detection. Method: Based on the advantages of CRISPR-Cas12a and recombinase polymerase amplification (RPA) technology, this experiment intended to establish an innovative technology suitable for rapid clinical detection of the L1 gene for HPV16 and develop a rapid nucleic acid lysis solution. The RPA technology was developed through primer verification to synthesize a large number of templates for the Cas12a-crRNA cleavage system swiftly. RPA technology was combined with lateral flow test strips to complete the interpretation of the results. The lysate contained 1.5 M guanidine hydrochloride, 50 mM Tris (pH = 8.0), 100 nM NaCl, 5 mM Ethylene Diamine Tetraacetic Acid (EDTA), and 1% Tween-20. Result: In this study, according to the concentration of the template and the results of agarose gel electrophoresis without dragging phenomenon, a third solution was finally determined as the optimal cleavage scheme: 1.5 M guanidine hydrochloride, 50 mM Tris (pH = 8.0), 100 nM NaCl, 5 mM EDTA, 1% Tween-20. After optimization, the optimal concentration of the probe was finally determined as 0.02 µM. Conclusions: The current study verified the rapid detetion capacity of CRISPR/Cas12a combined with RPA technology, hoping to provide a new technical method for HPV16 detection.