A Precise TaqMan-Based Real-Time qPCR Assay for Detecting and Quantifying Blackberry Chlorotic Ringspot Virus, Blueberry Shock Virus, and Plum Pox Virus in Fruit Tree Seedlings.

We developed a rapid and efficient TaqMan-based realtime reverse transcription quantitative PCR (RT-qPCR) assay for the detection and quantification of viruses infecting fruit trees, including blackberry chlorotic ringspot virus (BCRV), blueberry shock virus (BlShV), and plum pox virus (PPV). The detection limits for each virus were 40 copies (BCRV), 500 copies (BlShV), and 40 copies (PPV), respectively. Two primer-probe sets were selected for each virus, with amplification efficiencies ranging from 90-110%. High specificity was confirmed against other viruses or viroids sharing the same host plants. Multiplex detection of BCRV, BlShV, and PPV was achieved by using FAM and Cy5 fluorescent dyes. All sets maintained high efficiency and sensitivity with varying amounts of RNA extracted from the woody branches of the host plant. This assay will be useful for rapid and accurate diagnosis of plant virus diseases, especially in quarantine stations where leaf tissue is often unavailable upon import.