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

Authors: LIU, C - University Of California, Riverside; BODAGHI, S - University Of California, Riverside; Keremane, Manjunath; KALISH, B - University Of California, Riverside; VIDALAKIS, G - University Of California, Riverside; TSUTSUI, H - University Of California, Riverside

Submitted to: Advances in Sample Preparation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: One technique for diagnosis of virus diseases is LAMP (loop-mediated isothermal amplification), a variant of which is reverse transcripotion LAMP (RT-LAMP). LAMP is an isothermal (constant temperature) amplification technique for nucleic acids (NA). RT-LAMP combines this method of NA amplification with reverse transcription, making cDNA from RNA before amplification. This submission reports a sensing platform combining semi-automated sample preparation and RT-LAMP for the detection of citrus tristeza virus, an important RNA virus of citrus.

Technical Abstract: A sensing platform combining semi-automated sample preparation protocol and one-step reverse transcription loop-mediated sothermal amplification (RT-LAMP) is reported for rapid lorimetric detection of citrus tristeza virus (CTV) in a greenhouse. An OmniLyse microhomogenizer 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 ingreenhouse 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.