Development of multiplex primer and probe against tomato brown rugose fruit virus (ToBRFV), tomato spotted wilt virus (TSWV) and pepino mosaic virus (PepMV) disease factors in tomato and pepper plants

Tomato (Solanum lycopersicum) and pepper (Capsicum annuum) are indispensable vegetable species that have many different uses as human food in our country as well as in the world and contribute to the national economy. Virus diseases are one of the most important problems in tomato production. The inability to apply the chemical warfare method used against viruses and the fact that other control methods are not known by the manufacturer cause virus-related losses to increase. In this study, a Taqman® probe-based Real-time multiplex PCR method with three different primer-probe pairs was developed for the simultaneous detection of three important plant viruses. These viruses that cause a lot of yield loss in tomatoes and peppers are Tomato brown rugose fruit virus (ToBRFV), Tomato spotted wilt virus (TSWV) and Pepino mosaic virus (PepMV). The coat protein sequences of the three viruses mentioned above were obtained from the National Center for Biotechnology Information (NCBI) and BLAST was performed. Then, the common regions were determined and primer and probe designs were made using the Primer3 program. Since the main purpose of our Multiplex qRT-PCR study, which is a method that requires intensive optimization processes, is to provide a sensitive, fast and economical method, dilution series were prepared by determining the optimum conditions that will enable the PCR method to reach these goals. The FAM-channel was selected for ToBRFV, the HEX-channel for Tswv, and the Cy5-channel for Pepmv. The multiplex qRT-PCR experiment was successful with Ct values of 19.09, 21.89 and 21.63, respectively