Instrument-free detection of African swine fever virus in raw blood samples via CRISPR/Cas12a

Abstract

Nucleic acid amplification coupled with clustered regularly interspaced short palindromic repeats (CRISPR) detection has been widely applied in nucleic acid biosensing for its high sensitivity and specificity. However, the separated two-step increases the complexity of hands-on operations and the risks of amplicon-formed aerosol transmission, which hinders the popularization of nucleic acid detection for a wider range of scenarios. In this study, we propose a microchip-assisted detection platform for African swine fever virus (ASFV) detection. It involves ultrafast nucleic acid extraction, on-chip loop-mediated isothermal amplification (LAMP), CRISPR detection, and lateral flow assay-based visualized result readout. The liquid transfer between different reaction chambers of the microchip is driven by the gravity of liquid solutions. Therefore, complex liquid transfer and instruments are not required. The platform, which is named as All-In-Free (all nucleic acid detection steps included, instrument-free), can detect 10 copies/µL ASFV in approximately 45 min. For 32 real blood samples, the detection results of the All-In-Free platform are 96.3% positive and 100% negative consistent with polymerase chain reaction (PCR) testing. The All-In-Free detection platform has the advantages of low cost, high efficiency, and easy-to-operation, which is promising for popularization in low-resourced areas.