Development of a real-time RT-PCR assay for detection of tomato mottle mosaic virus

Tomato mottle mosaic virus (ToMMV) which is belonged to the tobamovirus genus was first identified in Mexico in 2013. According to the Turkish Ministry of Agriculture and Forestry's amendment on notification of emergency measures at World Trade Organizations (WTO), all countries are required to submit a Phytosanitary Certificate stating that the tomato and pepper seeds have been analyzed by the exporting countries using the RT-PCR method, indicating that the product is free of disease-causing agents such as ToMMV.To obtain a reliable and specific diagnostic method to detect the virus, new primers and probe set, targeting the conserved movement protein region of the ToMMV genome, was designed. In sillico assessment by BLAST analysis showed a 100% match to the all ToMMV isolates in NCBI database. After RNA extraction from ToMMV infected plant material the real-time amplification protocol was developed. The protocol was tested for analytical sensitivity, analytical specificity, repeatability and reproducibility obtaining consistent results for all of the validation parameters.This study is the validated real-time RT-PCR (reverse transcription PCR) detection method for ToMMV strains according to all the parameters required by the new diagnostic EPPO standard PM7/98 (4). This new protocol can be successfully applied at the Quarantine laboratories of the Turkish Ministry of Agriculture and Forestry for routine detection of ToMMV in pepper and tomato seeds.