qPCR ToBRFV set 192 (Tomato brown rugose fruit virus)

The qPCR ToBRFV set enables the detection of Tomato brown rugose fruit virus (ToBRFV) with a triplex real-time RT-PCR. Viral RNA of ToBRFV, extracted from tomato or pepper samples, is amplified with two distinct primer pairs in a one-step RT-PCR reaction. The amplification of the cDNA can be monitored in real time, because the specific probes are labeled with fluorophores (ToBRFV: FAM and HEX).

The target sequence for the FAM-probe and primer pair is located within the movement protein gene whereas the target sequence for the HEX-probe and primer pair is located within the coat protein gene of ToBRFV isolate Tom1-Jo. An internal positive control (IPC) is designed to detect a cytochrome oxidase (COX) gene sequence with a fluorophore-labeled (IC: ROX) specific probe. The control shows whether the extraction of tomato samples, the reverse transcription and the amplification of the RNA worked as intended. The qPCR ToBRFV set was developed and validated by BIOREBA.

The method is based on the primers of ISF-ISHI-Veg and was adapted from the diagnostic protocol referred to in the Appendix 4 of the PM 7/146 Tomato brown rugose fruit virus European and Mediterranean Plant Protection Organization (EPPO). Suitable tissue sources of viral RNA are tomato/pepper leaves, seeds and fruits. For the extraction of RNA, it is recommended to use standard RNA extraction protocols established for plant material. Upon request, BIOREBA can recommend an optimal extraction method.