A multiplex reverse transcription PCR assay for simultaneous detection of six main RNA viruses in tomato plants

Tomato virus diseases occur all around the world, causing serious yield losses. To detect these viruses quickly and provide a basis for disease control, a multiplex reverse transcription polymerase chain reaction system was established for simultaneous detection of Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato chlorosis virus (ToCV), Potato virus Y (PVY) and Potato virus X (PVX) in tomato plants, with 6 pairs of specific primers being designed based on the coat protein (CP) genes of these viruses. Transcriptional elongation factor-1α (EF-1α) from tomato was added to the multiplex RT-PCR reaction system to prevent false negatives. The concentration of the primers, annealing temperature, annealing time, extension time and amplification cycles were optimized. Expected fragments of 159 bp (ToCV), 262 bp (PVY), 362 bp (EF-1α), 430 bp (TMV), 500 bp (TSWV), 600 bp (CMV) and 705 bp (PVX) were amplified by this multiplex RT-PCR system, and their origin was confirmed by DNA sequencing. This method will have a wide application in virus detection of field samples.