Chain hybridization‐based CRISPR‐lateral flow assay enables accurate gene visual detection

Visualized gene detection based on the CRISPR‐Cas12/CRISPR‐Cas13 technology and lateral flow assay device (CRISPR‐LFA) has shown great potential in point‐of‐care testing sector. Current CRISPR‐LFA methodology mainly utilizes conventional immuno-based LFA test strips, which could visualize whether the reporter probe is trans-cleaved by Cas protein, indicating the target positive detection. However, conventional CRISPR‐LFA usually produces false-positive results in target negative assay. Herein, a nucleic acid Chain Hybridization‐based Lateral Flow Assay platform, named CHLFA, has been developed to achieve the CRISPR‐CHLFA concept. Different from the conventional CRISPR‐LFA, the proposed CRISPR‐CHLFA system was established based on the nucleic acid hybridization between the GNP‐probe embedded in test strips and ssDNA (or ssRNA) reporter from CRISPR (LbaCas12a or LbuCas13a) reaction, which eliminated the requirement of immunoreaction in conventional immuno-based LFA. The assay realized the detection of 1–10 copy of target gene per reaction within 50 min. The CRISPR-CHLFA system achieved highly accurate visual detection of target negative samples, thus overcoming the false-positive problem that often produced in assays using conventional CRISPR‐LFA. The CRISPR‐CHLFA platform was further adopted for the visual detection of marker gene from SASR‐CoV-2 Omicron variant and Mycobacterium tuberculosis (MTB), respectively, and 100% accuracy for the analysis of clinical specimens (45 SASR‐CoV-2 specimens and 20 MTB specimens) was obtained. The proposed CRISPR-CHLFA system could provide an alternative platform for the development of POCT biosensors and can be widely adopted in accurate and visualized gene detection.