Gross, A. J. | Search bioRxiv

bioRxiv2025

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

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

microbiology↗

bioRxiv2023

Degenerate PCR primers for potexvirus detection in seagrasses

2.Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity a?ects host-virus interactions. 3. Impact statementPotexviruses are widespread in terrestrial plants; however, the recent discovery of TVX in the seagrass Thalassia testudinum extends their host range to marine flowering plants. Here we use existing Potex-5 and Potex-2RC degenerate primers to explore potexvirus infections in several seagrass species. TVX sequence variants were detected in T. testudinum collected from the eastern Gulf of Mexico, uncovering previously unknown genetic diversity of this poorly understood virus. 4. Data summaryAll sequence data are available in NCBI GenBank under the accession numbers OR827692-OR827705, OR854648, OR863396, OR879052-OR879056, and PP430548-PP430571. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗