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• W4206603004 abstract "Recombinase polymerase amplification (RPA) has been recognized as a promising isothermal amplification method for nucleic acid detection. However, the digital format of RPA is still challenging to implement due to its MgOAc-initiated reaction feature and the inherent non-specific amplification. Here we develop a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) approach utilizing droplet microfluidics to achieve droplet digital RPA (ddRPA) for absolute nucleic acid quantification. By coupling a microfluidic picoinjector with a droplet generator, the reaction initiator MgOAc is dosed into droplets containing MgOAc-deprived RPA master mix for controlled digital reaction unlocking, which completely circumvents premature amplification. The discretization of the targets to a single-molecule level in confined droplets endows absolute quantification of the copy number. Coupled with CRISPR/Cas13a sensing, the ddRPA demonstrates single molecule detection ability within 30 min with significantly enhanced signal-to-noise ratio (S/N ratio>6) and uniform fluorescence signal reporting, facilitating the precise quantification of nucleic acids. Furthermore, the utility of the PADLOCK-CRISPR assay has been validated with 22 clinical samples, which generated results in 100% concordance with qPCR. We believe the coupling of droplet microfluidic technology with digital RPA will pave the way towards ultrasensitive and precise nucleic acid quantification." @default.
• W4206603004 created "2022-01-26" @default.
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• W4206603004 date "2022-04-01" @default.
• W4206603004 modified "2023-10-15" @default.
• W4206603004 title "Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection" @default.
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• W4206603004 doi "https://doi.org/10.1016/j.bios.2022.114019" @default.
• W4206603004 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/35078139" @default.
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