Journal: Animal Models and Experimental Medicine

Article Title: Development of RPA‐Cas12a ‐fluorescence assay for rapid and reliable detection of human bocavirus 1

doi: 10.1002/ame2.12298

Figure Lengend Snippet: (A) Schematic diagram of the RPA‐Cas12a‐fluorescence assay for HBoV1 detection based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas platform. The HBoV1 genomic DNA extracted from clinical samples was first pre‐amplified by RPA into double‐stranded DNA (dsDNA). In the Cas12‐mediated detection assay, the complex of pre‐amplified dsDNA, Cas12a and crRNA enables Cas12a activation, which then cuts the single‐stranded DNA molecules (ssDNA reporter). Finally, visualization of the RPA‐Cas12a‐fluorescence assay results is achieved by fluorescence and naked eye readouts. (B) and (C) Visualization of primers and probe for RPA (B) and crRNA spacer sites (C) within the target nucleoprotein 1 (NP1) gene of the HBoV1 genome. RPA primers, probe and crRNA are indicated by red, yellow and blue colored text, respectively.

Article Snippet: The conserved DNA fragments for the NP1 gene were produced by Sangon Biotech Co., Ltd., and cloned into the pBluescript II SK (+) plasmid to obtain pBluescript‐NP1. pBluscript‐NP1 was then transformed into Escherichia coli TOP10 for the production of recombinant pBluescript‐NP1 and stored at −80°C.

Techniques: Fluorescence, Recombinase Polymerase Amplification, CRISPR, Amplification, Detection Assay, Activation Assay