Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions.

Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaicvirus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significanteconomic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventingthe viruses from spreading and improving their control. In this study, a procedure based on twomultiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serialdilutions of positive controls were analysed by this methodology, and the results were compared withthose obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assayswere able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA.The multiplex versions were able to detect composite samples containing different concentrations ofspecific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected indifferent tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCRand commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, witha higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming thepresence of these seven virus species in Italy.