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

Authors: SUI, XUELIAN - Fujian Agricultural & Forestry University; ZHANG, SHOUAN - University Of Florida; WU, ZUJIAN - Fujian Agricultural & Forestry University; Ling, Kai-Shu

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2018
Publication Date: 1/12/2018
Citation: Sui, X., Zhang, S., Wu, Z., Ling, K. 2018. Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus. Journal of Virological Methods. 253:56-60. https://doi.org/10.1016/j.jviromet.2018.01.002.
DOI: https://doi.org/10.1016/j.jviromet.2018.01.002

Interpretive Summary: Tomato is one of the most important vegetables in the U.S., with an annual value well over $2 billion. Thrips-transmitted viruses are some of the most damaging viruses affecting tomato productions. Tomato spotted wilt virus (TSWV) is one of the most economically important plant viruses infecting tomato. In addition, the emerging Tomato chlorotic spot virus (TCSV) has resulted in serious crop losses in Florida in recent years, and has the potential to be spread to other parts of the world. Timely and accurate diagnosis is a prerequisite for effective disease management, but the current virus detection methods are not very useful in providing an accurate disease diagnosis. In the present study, ARS scientists in Charleston, SC, collaborated with other scientists in Florida and China and developed a simple and sensitive molecular detection method, called reverse transcription loop-mediated isothermal amplification (RT-LAMP), to achieve an effective species-specific detection of TCSV under field conditions. Further application of this newly developed technology for disease diagnosis will help to control TCSV on many economically important vegetables and ornamental plants and greatly benefit producers of these crops.

Technical Abstract: Tomato chlorotic spot virus (TCSV) is an emerging tospovirus that can cause severe disease on tomato plants. There are at least four tospoviruses infecting tomato, and mixed infection of various viruses in a field crop is quite common. With similarity in the symptomatology and cross serological reactivity among tomato-infecting tospoviruses, especially between TCSV and Groundnut ringspot virus (GRSV), the current serological tests could not achieve definite and accurate species-specific determination in disease diagnosis. To develop simple and sensitive species-specific detection, 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 other tospoviruses. These results demonstrate that this simple and sensitive RT-LAMP could be used to achieve species-specific detection for TCSV under field conditions.