Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus

Abstract

Tomato chlorotic spot orthotospovirus (TCSV) is an emerging orthotospovirus that can cause severe disease on tomato plants. There are at least four orthotospoviruses infecting tomato, and mixed infection of two or more orthotospoviruses in a single tomato plant is quite common in the field. With similarity in the symptomatology and cross serological reactivity among tomato-infecting orthotospoviruses, especially between TCSV and groundnut ringspot orthotospovirus (GRSV), the current serological tests could not achieve definite and accurate species-specific determination in disease diagnosis. Here, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for TCSV. Under optimum conditions, the virus was detected in as little as 0.2 ng of total RNA or in 1:10,000 dilution of a simple diluted tissue extract, which was ten times more sensitive than a conventional RT-PCR assay. The RT-LAMP assay was highly specific for TCSV, with no cross reaction with the other two orthotospoviruses: GRSV and tomato spotted wilt orthotospovirus (TSWV). These results demonstrate that this simple and sensitive RT-LAMP could be used to achieve species-specific detection for TCSV under field conditions.