POSTERS: Pathogen detection, quantification and diagnosis
Seed extract TaqMan RT-PCR for high throughput prescreening of ToBRFV in seeds of tomato
Harrie Koenraadt - Naktuinbouw. Debby Beugelsdijk- Enza Zaden Seed Operations BV, Sukhi Pannu- CSP Labs, Michel Ebskamp- Naktuinbouw, Carla Tavares- Enza Zaden Seed Operations BV, Gerbert Hiddink- Enza Zaden Seed Operations BV, Agata Jodlowska- Naktuinbouw, Maaike Bruinsma- Naktuinbouw
Tomato brown rugose fruit virus (ToBRFV) is a new Tobamovirus of tomato. Spread of ToBRFV by mechanical transmission is common, but potentially seed transmission is a pathway. ELISA and an indicator plant test are used to detect ToBRFV in seed extract, but do not discriminate between ToBRFV and more common tobamoviruses like ToMV and TMV. Two complementary ToBRFV-specific TaqMan RT-PCRs in a multiplex format were developed and implemented for seed health testing. One ToBRFV-infested tomato seed was diluted in healthy tomato seeds with variable subsample sizes. After grinding of seeds Bacopa chlorosis virus was spiked in the extract as a process control to monitor inhibition. RNA was purified on a Kingfisher platform. Finally, TaqMan RT-PCR was performed to detect presence of ToBRFV. Validation results of the ToBRFV seed extract (SE) TaqMan RT-PCR method will be presented. The SE TaqMan RT-PCR prescreening is high throughput, cost-effective and a specific method for ToBRFV in comparison with ELISA. A disadvantage of SE TaqMan RT-PCR and ELISA is that both methods cannot discriminate between infectious and non-infectious virus like the indicator plant test does. This SE TaqMan RT-PCR is a reliable prescreening method for ToBRFV and is complementary to the reference indicator plant test that distinguishes between infectious and non-infectious ToBRFV among other relevant tobamoviruses.