Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: Tomato necrotic stunt virus

• Offered a high sensitive detection that was comparable to that of a conventional RT-PCR.
• Able to detect the presence of ToNStV in tomato and a broad range of solanaceous plant species using either a purified total plant RNA or diluted crude tissue extract.
• Complemented our previous reported RT-PCR and real-time RT-PCR assays in ToNStV detection.
• Highly specific and had no cross-reaction to other potyviruses (PVY and TEV) and several common tomato infecting viruses and viroids.
• Could provide a simple, rapid, accurate, and sensitive field diagnostic method for ToNStV.

Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for 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 with 1:20,000 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 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 RT-PCR and real-time RT-PCR assays reported previously, with a potential to provide a simple, rapid, and sensitive field diagnostic method for ToNStV.