Rapid colorimetric detection of Citrus tristeza virus combining portable sample preparation and reverse transcription-loop mediated isothermal amplification

This article details combined portable sample preparation and colorimetric detection using loop-mediated isothermal amplification (RT-LAMP) of Citrus tristeza virus (CTV) from citrus leaves. The sample preparation employs an OmniLyse micro-homogenizer and cellulose paper disks to extract and isolate total nucleic acids in <15 min. Primer and dimethyl sulfoxide (DMSO) concentrations were optimized to minimize RT-LAMP assay reaction times and maximize the delay in the appearance of false positives due to non-specific amplification, respectively. The CTV primers were assessed against a panel of 24 CTV-positive isolates and 6 non-CTV pathogen isolates. To adapt the protocol for cold-chain-free field deployment, a lyophilized RT-LAMP reagent mix was developed and its rehydration solution was optimized to minimize false positives. In a greenhouse setting, the micro-homogenizer successfully extracted and isolated nucleic acids from CTV-positive trees, followed by the lyophilized RT-LAMP assay positively detecting the pathogen within 35 min. This study establishes the feasibility of quick and portable CTV detection without access to laboratory equipment, paving the way for larger-scale field studies, comprehensive validation of assay performance, and potential extension to other plant pathogens.