Simultaneous detection of protein and nucleic acid biomarkers with a CRISPR-based assay

Simultaneous analysis of multiple protein and nucleic acid biomarkers in a single clinical sample can yield clinically valuable results for diagnosing, surveillance, and managing various diseases. However, the current diagnostic platforms depend largely on protein- or nucleic acid-independent assay, which is time-consuming and laborious. Here, we propose a novel assay termed MCD assay that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene RNA and SARS-CoV-2 S protein. This assay was based on magnetic separation technology, combined with the clustered regularly interspaced palindromic repeats (CRISPR) /CRISPR-associated proteins (Cas) system for accessible point-of-care detection of nucleic acids and proteins. The assay could recognize target molecules through nucleic acid hybridization or antigen-antibody reactions and integrated with the CRISPR/Cas-based enzymatic detection; the lateral flow test strips enabled visualization of the assay results. The limit of detection of MCD was 1000 copies/mu L for viral nucleic acid and 10 pg/mL for viral protein. The combined evaluation of multiple biomarkers from diverse pathways can enhance the accuracy and specificity of disease diagnosis. It is also crucial to use a single platform for detecting biomarkers across various dimensions to evaluate and manage major diseases.