Research on PER Signal Amplification Method Based on CRISPR Detection System

WANG Xiaojun; ZHANG Yiqing; TAN Ya; WANG Jichuang; CHENG Lei; WANG Xudong; WANG Yunlong

doi:10.3969/j.issn.1004-437X.2025.05.002

Abstract：Objective To establish a CRISPR/Cas13a-based detection signal amplification method for novel coronaviruses,namely CRISPR/PER,using a spacer-associated short palindromic repeat-associated Cas13a protein(CRISPR-Cas13a)in combination with a cascade primer exchange reaction(PER).Methods Screening of the novel coronavirus template strand and design of two specific CRISPR RNAs(crRNA)for the template strand,optimisation of the CRISPR reaction system using the cleavage activity of Cas13a protein,screening of the paired hairpin structure and primers for the PER reaction,optimisation of the PER reaction system using the screened pairs and design of specific probes P2 probe and the P3 probe were designed,and the signal probe P3 was combined with the amplified hairpin H27 using base complementary pairing to form the reaction solution,and colloidal gold test strips were prepared.Results The best paired crRNA for RNA was crRNA-1,500 nmol·L-1 Cas13a protein added to 1 μL,4 g·L-1 of crRNA added to 0.5 μL and 5 U Ribonuclease Inhibitor added to 1 μL were the optimal addition amounts for the CRISPR reaction system.The H27 hairpin structure paired best with the P27 primer.the optimum conditions were 37 ℃ and 40 minutes reaction time.The optimum addition amounts of each component were 1 μL of 4 U Bst DNA polymerase,10 μL of 5 μmol·L-1 H27 hairpin,1 μL of 100 mmol·L-1 MgSO4 and 2 μL of dNTP,respectively.The minimum detection limit of the novel coronavirus template was 100 μg·L-1,and the specificity test showed no cross-reactivity with the three control viruses.Conclusion The CRISPR/PER assay is convenient,rapid and specific for the detection of novel coronaviruses,and can be used as a reference for the investigation of new nucleic acid detection methods.