Protocols

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ToBRFV PROTOCOLS

Protocols B, C, and D are diagnostic protocols developed and implemented for the detection of the Tomato Brown Rugose Fruit Virus (ToBRFV) in tomato and pepper seeds. These protocols are considered optimal for phytosanitary testing by the National Plant Protection Organizations (NPPOs) of North American Plant Protection Organization (NAPPO) region (Mavrodieva et. al., 2025).

The following information is available regarding these protocols:

  • Optimal Performance: These protocols perform optimally across all tested parameters and variables and are recommended for phytosanitary testing.
  • Harmonization and Trade: Their use by NAPPO member countries can streamline trade by potentially removing the need for retesting at borders, thus easing the movement of tomato and pepper seeds.
  • Methodologies: Protocols B, C, and D use Real-time RT-PCR and conventional PCR methodologies. These methods are sensitive and automatable for detecting ToBRFV.
  • Specific Protocols:
    • Protocol B is the ISHI-Veg/NSHS protocol.
    • Protocol C (Multiplex) was developed by the USDA-APHIS (Chanda et al., 2021).
    • Protocol D (Conventional RT-PCR) was also developed by the USDA-APHIS (Dey et al., 2021).

Reference: Mavrodieva et. al., 2025. Pilot for Harmonization of Diagnostic Protocols for Tomato Brown Rugose Fruit Virus (ToBRFV) in Tomato and Pepper Seeds. PhytoFrontiers 5: 187-196.