A simple and rapid diagnostic method for 13 types of high-risk human papillomavirus (HR-HPV) detection using CRISPR-Cas12a technology

Jiaojiao Gong,Qianyun Li,Menghao Liu,Liping Liang,Liang Xue,Guanghui Zhang,Guanghui Tang,Wangguo Wang

doi:10.1038/s41598-021-92329-2

Abstract

Cervical carcinoma is the second most common cancer in women worldwide with greater than 99% of the cases caused by human papillomaviruses (HPVs). Early detection of HPVs especially the high risk types (HR-HPVs) are essential to prevent the disease progression. The existing methods for HPV detection, such as qPCR are of high sensitivity and specificity, but the need for expensive machinery and well-trained personnel slow down the disease detection. The emerging Cas12a-based method presents a new technique for nucleic acid detection. However, it is time-consuming and labor-intensive when used for HPV detection, as several reactions are required in order to identify multiple HPV infections. We herein present a non-genotyping method for 13 types of HR-HPV detection in a single reaction by combining the isothermal recombinase polymerase amplification (RPA) method with CRISPR-Cas12a technology. The result could be achieved in 35 min with high sensitivity (500 copies per reaction). This assay represents great advances for the application of RPA-Cas12a system and holds a great potential to address the key challenges facing the HPV diagnostics.

Highlights

Human papillomavirus (HPV) infection is a major causative agent of cervical cancer in w omen[1]
According to the guidelines for nucleic acid testing of HPV released by Chinese Food and Drug Administration (CFDA) in 2015, 13 types of high risk (HR)-HPV are required to be identified when developing a diagnostic assay
The kinetic fluorescence curve of Cas12a reaction showed that only a few HPV types generated strong signal, indicating weak amplification for most HPV types with this primer pool (Fig. S1)

Summary

Introduction

Human papillomavirus (HPV) infection is a major causative agent of cervical cancer in w omen[1]. Our assay consists of an RPA amplification using the primer pool derived from PGMY/GP6+ primer set, and a detection mediated by CRISPR-Cas12a (Fig. 1). The kinetic fluorescence curve of Cas12a reaction showed that only a few HPV types generated strong signal, indicating weak amplification for most HPV types with this primer pool (Fig. S1).

Results

Conclusion