Simultaneous detection of stone fruit tree viruses by one-step multiplex RT-PCR

A protocol of multiplex RT-PCR in a one-tube system for the detection of the most common stone fruit trees viruses [e.g., plum pox virus (PPV), prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV)], including the internal control of NADH dehydrogenase subunit 5 (nad5) gene are described here. The method specificity was tested on more than 80 different samples with various isolates and strains of the viruses. It showed that the targeted viruses produced the expected amplicons, whereas all other related viruses produced only the nad5 internal control amplicon. The method sensitivity was evaluated by comparing it with Simplex RT-PCR with the same primers; no significant differences in detection limits were recorded. Furthermore, the competitiveness of the primers in the assay was tested by serial RNA dilutions of samples with mixed and single infections. The least competitive was the internal control nad5 gene primer pair; therefore, there is a reduced risk of false negatives as all the other primers tend to be more efficient in the given primer cocktail than in the primers for internal control.