A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid colorimetric detection of pepper mild mottle virus (PMMoV)

Authors: GROSS, ANTHONY - University Of South Florida; Lopez Jr, Salvador; ROGERS, ALEXANDRA - University Of South Florida; Adkins, Scott; BREITBART, MYA - University Of South Florida

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2025
Publication Date: 7/17/2025
Citation: Gross, A.J., Lopez Jr, S., Rogers, A., Adkins, S.T., Breitbart, M. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid colorimetric detection of pepper mild mottle virus (PMMoV). Journal of Virological Methods. 338:115225. 2025. https://doi.org/10.1016/j.jviromet.2025.115225.
DOI: https://doi.org/10.1016/j.jviromet.2025.115225

Interpretive Summary: Pepper mild mottle virus (PMMoV) is a significant pathogen of pepper and related agricultural crops worldwide. Accurate diagnostic tests are required for its identification, to allow rapid management decisions to be made. In this report, we describe development and validation of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for PMMoV for in-field diagnosis of this virus. This approach will be applicable for PMMoV detection for growers, Extension personnel, and local and Federal regulatory and research scientists.

Technical Abstract: Pepper mild mottle virus (Tobamovirus capsica, PMMoV) is a plant virus in the genus Tobamovirus that infects peppers and other members of the family Solanaceae. The virus is transmitted mechanically, poses a significant threat to crops globally, and is one of the most abundant viruses found in human feces and wastewater. Two colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect PMMoV, one targeting the RNA-dependent RNA-polymerase (PMMoV_RdRp) and one targeting the coat protein (PMMoV_CP). Synthetic gBlock positive controls were used to determine each assay’s detection limit. PMMoV_RdRp detected PMMoV at concentrations greater than or equal to 100 copies/µL, the same sensitivity as the corresponding RT-PCR assay. In contrast, the detection limit of the PMMoV_CP RT-LAMP assay was an order of magnitude greater. Both assays were specific to PMMoV and did not amplify plant host tissue or related tobamoviruses. Since these RT-LAMP assays do not require specialized laboratory equipment and yield positive results within 20-30 minutes, they are advantageous for point-of-use testing. Overall, the RT-LAMP assays described here are sensitive, specific, and faster than existing methods for PMMoV detection and quantification and thus have potential widespread applications for agriculture, wastewater treatment assessment, recreational water quality testing, and food safety.