RESUMO
High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.
Assuntos
Microfluídica , Ácidos Nucleicos , Bioensaio , Sistemas CRISPR-Cas , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismoRESUMO
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.