Multiplex PCR methods for simultaneous detection of tomato brown rugose fruit virus, tomato spotted wilt virus and pepino mosaic virus

Tomato (Solanum lycopersicum) is a crucial vegetable globally, pivotal in securing the world's food supply. However, viral diseases pose a significant threat to tomato cultivation, resulting in considerable yield losses. Employing sensitive, rapid, and cost-effective detection methods is imperative for mitigating such losses in agricultural production. In response, we developed a Taqman® probe-based real-time multiplex PCR method capable of concurrently detecting three major plant viruses: Tomato brown rugose fruit virus (ToBRFV), tomato spotted wilt orthotospovirus (TSWV), and pepino mosaic virus (PepMV), all of which are prominent viral pathogens affecting tomato production. Utilizing envelope protein encoding sequences of these pathogens, we designed primers and probes, assigning the FAM reporter for ToBRFV, the HEX reporter for TSWV, and the Cy5 reporter for PepMV detection. The multiplex RT-qPCR experiment yielded successful results, with Ct values of 29.34, 25.93, and 27.47 for ToBRFV (at 10⁻⁶ dilution), TSWV (at 10⁻² dilution), and PepMV (at 10⁻³ dilution), respectively. Developed primers facilitate the early detection of destructive pathogens ToBRFV, TSWV, and PepMV using both RT-PCR and RT-qPCR, offering a user-friendly and cost-effective approach.