Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR

A pentaplex reverse-transcription polymerase chain reaction (Pentaplex RT-PCR) in a single tube was developed for the simultaneous detection of the pome fruit viruses: Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). This is the first report of the simultaneous detection of all four viruses and host mRNA as an internal specific control. Pentaplex RT-PCR was applied successfully throughout the year, using different plant organs (leaves or dormant buds). The sensitivity of detection by monoplex- and pentaplex RT-PCR assays was comparable. Different combinations of mixed infections of viruses were identified in samples of infected apple and pear trees from different geographical regions. The pentaplex RT-PCR assay developed was sensitive, simple, rapid, and reliable for simultaneous detection of the four viruses in extracts of leaves or dormant buds.