Development of a two-step RT-multiplex PCR assay for the simultaneous detection of six viruses with a wide host range, including fruit-tree, for use in post-entry plant quarantine inspections in Japan.

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Viruses cause significant economic losses to fruit-tree orchards by reducing fruit yield and quality. Among viruses that infect grapevines ( spp.) and prunuses ( spp.), carnation ringspot virus, peach rosette mosaic virus, raspberry ringspot virus, strawberry latent ringspot virus, sowbane mosaic virus, and grapevine vein yellow virus (tomato ringspot virus) have all been designated as plant quarantine pathogens in Japan. Although these viruses can be screened using sap inoculation on quinoa (), it is difficult to identify the species based solely on symptoms. Several diagnostic tests can be applied to diagnose viral infections in plants; however, by and large, polymerase chain reaction (PCR) is the most commonly used method. In particular, multiplex PCR allows simultaneous detection of multiple targets in a single assay, thereby reducing costs, labor, and time. Therefore, reliable diagnostic methods using PCR based on the genetic diversity of viruses are critical for detecting viral infections in fruit-tree orchards. In this study, we developed a two-step reverse transcription (RT)-multiplex PCR for quick and cost-effective detection of the six viruses listed above in infected quinoa, using newly designed primer sets. Primers were designed for each viral variant based on all sequence data obtained from the NCBI database. The detection sensitivities of our assay were equivalent to or even 1-10,000 times greater than those of previously reported singleplex RT-PCR assays, with the added advantage of zero non-specific reactions occurring. The proposed assay will be useful for identifying and selecting healthy nurseries and for plant quarantine inspections.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s13337-025-00911-3.