DETECTION AND ABSOLUTE QUANTIFICATION OF PINEAPPLE MEALYBUG WILT-ASSOCIATED VIRUS-2 IN PINEAPPLE USING REAL-TIME RT-PCR (TAQMAN^®) ASSAYS

A reliable, rapid, and sensitive real-time RT-PCR (qRT-PCR) assay using DyNAmo^® TaqMan^® RT-PCR was developed to detect and quantify PMWaV-2 in pineapple plants. Oligonucleotides used for the assay were designed from the coat protein regions of PMWaV-2. Absolute quantification of PMWaV-2 was achieved by generating standard curves from RNA transcripts cloned from the selected target of the PMWaV-2 CP gene. PMWaV-2 concentrations were higher in the white basal portion compared to other parts of the leaves. PMWaV-2 levels were higher in younger leaves compared to older leaves on the same plant. Virus titres were significantly higher in PMWaV-2-infected pineapple roots compared to the leaves. PMWaV-2 infected plants with mealybug wilt of pineapple (MWP) symptoms had higher virus levels than those without wilting symptoms. And lastly, pineapple virus titres were significantly affected by the presence or absence of viruliferous mealybugs on PMWaV-2 infected plants.