Journal: Bioactive Materials
Article Title: An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip
Figure Lengend Snippet: Detecting N gene of SARS-CoV-2 with a onepot method using phosphorothioate modified primers. a , Representative plot of fluorescence intensity versus time for onepot detection of N gene of SARS-CoV-2 plasmid using three unmodified-primer pairs (left panel). Fluorescent signal was obtained at 30 min after reaction (right panel). b , Primers were modified with phosphorothioate on the first two phosphate backbones proximity to 5′ and 3′ end. crRNA was designed to have two nucleotides overlapping with the reverse primer (upper panel). Modified F: forward primer modified with phosphorothioate; modified R: reverse primer modified with phosphorothioate. c , Intact amplicons derived from the modified primers (left panel) and nicked dsDNA products after Cas12a cis cleavage (right panel). d , Schematic of TESTOR workflow. SSB, single-stranded DNA binding protein; F, fluorophore; Q, quencher. e , Real-time fluorescence detection of the TESTOR assay for N gene of SARS-CoV-2 (N0 region) and 10 5 copies of plasmid DNA was used. f , Fluorescence kinetics of two primer pairs for N gene of SARS-CoV-2 (N0 region) detection (left panel) in a closed-tube. Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. g , Analytical sensitivity of TESTOR for N gene of SARS-CoV-2 (N0 region) detection (left panel). Fluorescent signal was measured at 30 min after reaction (right panel) using 10 5 copies of plasmid DNA. h , Another region of N gene of SARS-CoV-2 (N1 region) was detected using 10 5 copies of plasmid DNA template. i , Analytical sensitivity of TESTOR for N1 gene of SARS-CoV-2 detection. Signals were obtained using a plate reader in an uncapped 96-well plate ( a , e ) or using an real-time PCR detection system in a capped PCR tube ( f, g, h, i ). Error bars represent the mean ± s.d., where n = 3 replicates ( a, f, g, h, i ). ***, P
Article Snippet: Wang et al. added Cas12a on the inner wall of the reaction tube, and a centrifugation step was followed to initiate the cis - and trans -cleavage of Cas12a after RPA reaction [ ].
Techniques: Modification, Fluorescence, Plasmid Preparation, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction