Advances in the integration of isothermal amplification with CRISPR-Cas systems for point-of-care nucleic acid biosensing in viral, environmental, and food safety applications
Chenyang Song, Chuanjin CuiPurpose
This study aims to systematically examine the integration of isothermal amplification (IA) with CRISPR-Cas systems for point-of-care nucleic acid biosensing, elucidating the synergistic mechanisms that resolve the sensitivity–specificity tradeoff in molecular diagnostics.
Design/methodology/approach
A comprehensive literature survey (2017–2026) compares one-pot vs. two-tube assay configurations, evaluates key parameters of LAMP/RPA coupled with Cas12/Cas13, and critically assesses fluorescence, colorimetric lateral flow and SERS signal readouts.
Findings
IA-CRISPR enables rapid (≤ 30 min) and attomolar-sensitive detection. One-pot assays mitigate aerosol contamination and operational complexity; two-tube formats remain valuable for validation. Applications cover viral pathogens (SARS-CoV-2, Ebola, dengue, HBV, HPV), environmental surveillance (invasive species, harmful algal blooms, tetracycline) and food safety (hygiene indicators, Salmonella). Persistent challenges include intrinsic cleavage–amplification incompatibility in single-tube reactions, labor-intensive primer/crRNA design, nascent microfluidic integration and lack of standardization.
Originality/value
This review provides a holistic, multidimensional framework that tracks technological evolution, appraises assay strategies and identifies four future directions – engineered conditionally-activated Cas variants, AI-driven design pipelines, microfluidic/paper-based device integration and harmonized performance standards – offering a concrete roadmap to bridge proof-of-concept research and commercially viable decentralized diagnostics.