Atallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I., and Stevenson, W.R.
Notes: These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays. (3673)