Colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and specific detection of sunflower chlorotic mottle virus in sunflower

Abstract

Sunflower chlorotic mottle virus (SuCMoV), an emerging sunflower-infecting virus in Thailand, was considered as one of the most frequently emerged viruses infecting sunflowers causing chlorotic spots, mottle, yellowing and stunting symptoms. This study aimed to develop and validate the colorimetric one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of SuCMoV in sunflowers. We optimized the RT-LAMP conditions and determined that incubation at 65 °C for 30 min in a heat block incubator provided optimal results, as confirmed by the presence of ladder-like pattern LAMP products visualized through agarose gel electrophoresis. In the colorimetric detection system, positive reactions were clearly indicated by a color change from pink to yellow, while negative reactions remained pink. The specificity test verified that the LAMP primers did not cross-react with other potyviruses. Sensitivity analysis demonstrated that the RT-LAMP assay could detect SuCMoV RNA at concentrations as low as 10 fg/μL which were greater than those of one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) and one-step RT-PCR. Validation of the colorimetric one-step RT-LAMP assay using naturally infected sunflower samples confirmed its ability to successfully detect SuCMoV, even in mixed viral infections with closely related sunflower-infecting potyviruses detecting by multiplex RT-PCR. To our knowledge, this study represents the first development of a colorimetric one-step RT-LAMP assay for rapid and sensitive detection of SuCMoV.