SCAN: A sample-to-answer cross-priming isothermal assay for on-site virus detection with RT-qPCR sensitivity and genomically similar virus differentiation specificity.

Abstract

Genomically similar viruses often differ in pathogenicity and host tropism due to specific mutations, and failure to distinguish them risks misdiagnosis and ineffective control. Molecular methods can differentiate such viruses but require laboratory settings and skilled personnel, while field-deployable immunological methods suffer from cross-reactivity. To address this challenge, we developed SCAN (Sample-to-answer Cross-priming isothermal amplification Assay with Nucleic acid strip), a general framework for on-site detection of genomically similar viruses. Comparative bioinformatics of isolation and sequencing data identifies key conserved differential determinants for primer design, ensuring specificity and reducing non-specific amplification. A one-tube cross-priming isothermal amplification (CPA) enables rapid target amplification without thermal cycling, and the products are visually detected on a nucleic acid strip. All steps are integrated into a handheld, lightweight device (9.9&#x202f;&#xd7;&#x202f;4.4&#x202f;&#xd7;&#x202f;3.3&#x202f;cm, <200&#x202f;g) that also prevents aerosol contamination. Using transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), the latter a natural mutant of TGEV, as a model, SCAN achieves a detection limit of 102 copies/&#x3bc;L with sensitivity comparable to RT-qPCR and supports sample-to-answer testing within 80&#x202f;min and simple operations. With verified high sensitivity, specificity, and accuracy, as well as field usability, SCAN provides a generalizable route for developing point-of-care tests (PoCT) that require precise field differentiation of closely related pathogens.