A real-time, fluorescent, reverse transcription-polymerase chain reaction(RT-PCR) assay was developed for the detection of Plum pox potyvirus (PPV). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by electrophoretic visualization with ethidium-bromide. All strains of PPV were detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantification of viral template. The assay was used to measure the levels of virus acquired by several aphid species.