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

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 the other targeting the coat protein (PMMoV_CP). Synthetic gBlock positive controls were used to determine the detection limit of each assay. PMMoV_RdRp detected PMMoV at concentrations greater than or equal to 100 copies/L, the same sensitivity as the RT-qPCR assay for this gene. 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 more rapid 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. HighlightsO_LIRT-LAMP assays were developed for the detection of the PMMoV RdRp and CP genes C_LIO_LIThe RdRp assay matches the detection limit of the established PMMoV RT-qPCR assay C_LIO_LIPositive results are obtained within 20-30 minutes from the reaction start C_LIO_LIThese RT-LAMP assays are specific to PMMoV and do not amplify related viruses C_LIO_LIThese assays are applicable for PMMoV detection across diverse scientific fields C_LI