HPV 16 和 18 基因分型 PCR-LbCas12a 检测方法

Abstract

CRISPR/Cas12a systems have been widely used in nucleic acid detection, because of their high specificity and trans-cleavage activity. We combined the specificity of CRISPR/Cas12a system with the sensitivity of PCR, to develop a new HPV detection assay with high specificity and sensitivity, called PCR-LbCas12a assay. Here, we pre-amplified almost all high-risk HPV genotypes with consensus primer (MY09/11) and added to new tubes containing HPV 16 or 18 CRISPR/Cas12a system for HPV genotyping. The LOD of PCR-LbCas12a for HPV16 or HPV18 DNA was 100 copies and 10 copies per reaction, respectively. In mixed samples of HPV 16 and HPV 18 DNA, PCR-LbCas12a assay could detect HPV16 DNA from samples containing 10 2 copies of HPV16 and 10 4 copies of HPV18. On the other hand, HPV18 DNA was detected from samples containing 10 3 copies of HPV16 and HPV18, respectively. Following validation with HPV-positive and HPV-negative cell lines (SiHa, HeLa, and C-33A), the PCR-LbCas12a assay was evaluated using 39 clinical samples previously characterized by type-specific qPCR. The assay demonstrated a sensitivity of 91.3 (95 CI 71.9–98.9) and a specificity of 100. The PCR-LbCas12a assay can discriminate the HPV 16 and HPV 18 genotypes, and holds great potential in simultaneous detection of most high-risk HPV genotypes including HPV 16 and HPV18.