Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus

Abstract

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.