Location: Crop Improvement and Protection Research
Project Number: 2038-22000-020-003-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 30, 2021
End Date: Jul 29, 2023
Objective:
Objective 1: Develop a multiplex real-time RT-PCR for screening detection of tomato torrado virus (ToTV) in plants and seeds to include internal plant control.
Objective 2: Develop a conventional RT-PCR for confirmatory detection of ToTV in plants and seeds matrices to be combined with sequencing for identification.
Objective 3: Develop a multiplex real-time RT-PCR for detection of several torradoviruses infecting tomato.
Approach:
Primers and probes will be developed to known sequences of ToTV and existing tomato infecting torradoviruses. For the ToTV multiplex real-time RT-PCR, two primer/probe sets to target y capsid (coat) protein (CP) and RNA-dependent RNA Polymerase (RdRp) will be selected and evaluated. For the ToTV conventional RT-PCR, primer sets will be developed to the most characteristic gene sequences of size that provide enough sensitivity and specificity.
Primers and probes will be tested independently to verify that each target is amplified specifically and efficiently, and that non-target viruses and host plant nucleic acids do not amplify. Each primer and probe will subsequently be evaluated as a part of a specific multiplex system.
