Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2014
Publication Date: 2/3/2014
Citation: Li, R., Ling, K. 2014. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus. Journal of Virological Methods. 200:35-40.
Interpretive Summary: Tomato necrotic stunt virus (ToNStV), a new virus infecting tomato plants in Mexico, was recently identified in our laboratory. The current detection method requires very expensive purified sample preparation, a sophisticated thermal cycling instrument, and a highly skilled technician. In the present study, ARS scientists in Charleston, South Carolina developed a simple and sensitive detection assay that could provide a reliable field diagnosis for this emerging viral disease. This new technique can be readily applied without highly skilled and technical expertise, allowing for quick diagnosis of the problem virus. This method is of great interest to producers and diagnostic companies who need effective and inexpensive methods to identify damaging disese agents.
Technical Abstract: Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to that of conventional RT-PCR, with detection of ToNStV in a reaction containing only 8 pg of total tomato RNA or with1:20,000 dilution of crude tissue extract. This assay was able to detect ToNStV in a broad range of solanaceous plant species. The RT-LAMP for ToNStV was highly specific with no cross-reactivity to other potyviruses (i.e. Potato virus Y and Tobacco etch virus), as well as several other common tomato viruses. RT-LAMP should complement previously reported RT-PCR and real-time RT-PCR assays with a potential to provide a simple, rapid, and sensitive field diagnostic method for ToNStV.