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bioRxiv · 10.1101/2024.11.09.622765

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

Liu, C.-W.·Bodaghi, S.·Keremane, M. L.·Kalish, B.·Vidalakis, G.·Tsutsui, H.
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Abstract

AbstractsA 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/L (equivalent to 86 copies/mg of tissue) and 5 copies/L (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.

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Liu, C.-W., Bodaghi, S., Keremane, M. L., Kalish, B., Vidalakis, G., Tsutsui, H.. 2024-11-11. Rapid colorimetric detection of citrus tristeza virus combining portable sample preparation and reverse transcription-loop mediated isothermal amplification. https://doi.org/10.1101/2024.11.09.622765

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