Development and verification of a one-step RT-PCR method to diagnose tomato brown rugose fruit virus in Solanaceae cultivation in Japan

Abstract

Tomato brown rugose fruit virus (ToBRFV) is a Solanaceae-infecting tobamovirus and spreading rapidly worldwide. Although ToBRFV has not been reported on any crops in Japan, simple and specific detection methods of ToBRFV that can distinguish it from other tobamoviruses such as tomato mosaic virus (ToMV) are urgently required to prepare for possible occurrences of ToBRFV in Japan. In this study, we developed a detection method that combined newly designed primers that are highly specific to ToBRFV with a one-step conventional RT-PCR. The primers were designed within the conserved regions of the coat protein gene of ToBRFV to distinguish it from other tobamoviruses. This method produced distinct positives in tests of ToBRFV-infected leaves of tomato, bell pepper, and eggplant and negatives in tests of healthy leaves or leaves infected with six other Solanaceae-infecting tobamoviruses in Japan. Furthermore, this method was applicable to a wide range of concentrations of total RNA from leaf samples and leaves with very low virus concentrations. Thus, this method would be suitable for primary and early detection of ToBRFV when ToBRFV-like symptoms were observed in solanaceous crops in Japan.