Rapid colorimetric detection ofcitrus tristeza viruscombining portable sample preparation and reverse transcription-loop mediated isothermal amplification

Abstract

A sensing platform combining semi-automated sample preparation protocol and one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) is reported for rapid colorimetric detection of Citrus tristeza virus (CTV) in a greenhouse. An OmniLyse micro-homogenizer and cellulose paper disks were integrated for quick sample preparation of total nucleic acids (<15 min). RT-LAMP assays were optimized in terms of primers' concentrations and minimization of false positives for both CTV and cytochrome oxidase (COX) detections. Specifically, the optimal reaction time for lab-based RT-LAMP assays was determined as 40 minutes with the detection limits of CTV and COX as 43 copies/μL (equivalent to 86 copies/mg of tissue) and 5 copies/μL (equivalent to 10 copies/mg of tissue), respectively. Additionally, an in-greenhouse colorimetric RT-LAMP assay with lyophilized reaction mix for endpoint CTV detection was successfully conducted in 35 minutes without a false response in either colorimetric or fluorometric assays. Overall, this quick sample preparation protocol integrated with the lyophilized RT-LAMP assays showed high efficiency and reliability in plant pathogen detection in a greenhouse. This strategy holds great potential to be integrated into a portable, autonomous system and be universally adopted for in-field diagnosis of different pathogens.