Detection and absolute quantitation of watermelon mosaic virus by real-time RT-PCR with a TaqMan probe

Watermelon mosaic virus (WMV) causes serious damage to several crops worldwide, mainly cucurbits. Disease control is based on preventing spread and search for natural resistances for plant breeding, which requires tools for sensitive detection and precise quantitation. We developed a procedure based on reverse transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR) with a primer pair and a TaqMan (R) probe specific for WMV. The primers and probe were designed from conserved sequence stretches to target a wide range of WMV isolates. A standard curve performed with transcripts enabled estimation of WMV RNA copies per ng of total RNA, with a wide dynamic range and sensitivity (10(4) to 10(11)). This RT-qPCR was assayed with field samples from different cucurbits and used to evaluate the temporal accumulation in pumpkin plants.