Development of virus detection by RT-PCR for tomato brown rugose fruit virus in commercial tomato and pepper seed in Thailand

Abstract

Tomato brown rugose fruit virus (ToBRFV) is a quarantine pest, which infected in tomato and pepper seeds. The surveillance and phytosanitary measures in Thailand require inspection of imported or exported seeds to ensure that ToBRFV is not detected. Detection method for ToBRFV in tomato and pepper seeds by molecular techniques was carried out. A pMA-RQ-REF recombinant plasmid encompassing TOBRFV replicase gene of 560 bp was amplified and subsequently transformed into Escherichia coli strain DH5a. Six transformants were randomly selected and analyzed. The result showed that all of them contained the insert with 100% identity to those reported in the GenBank. Since there have been no report of this virus in Thailand, therefore total RNA was extracted from healthy pepper and tomato seeds for cDNA synthesis. The pMA-RQ-REF recombinant plasmid was spiked into the cDNA for positive control preparation. Detection method was performed using reverse transcription-polymerase chain reaction (RT-PCR) which the PCR product at 560 bp was obtained. The developed method was highly specific to ToBRFV without cross reaction to other 13 virus species in 5 genera. Sensitivity test for TOBRFV detection was conducted by diluting the amplified replicase gene at 100 ng/µl in deionized water, pepper and tomato seed sap. The result show a sensitivity at 1x10^-6 pg/µl and 100 pg/µl, respectively. Evaluation of the developed method by testing commercial seeds including 71 tomato samples and 174 pepper samples showed negative results to those performed by Plant Quarantine Research Group, Plant Protection Research and Development Office, Department of Agriculture with Real-time RT-PCR technique.