Real-time assay for quantitative detection of non-persistently transmitted Plum pox virus RNA targets in single aphids
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A TaqMan real-time RT-PCR was developed to detect and quantify RNA-targets from the non-circulative, non-persistently transmitted Plum pox virus (PPV) in individual fresh or aphids captured previously and squashed on paper. Reliable quantitation ranged from 40 up to 4 x 10(8) copies of control transcripts. This technique was applied successfully to plant material and to individual PPV vector (Myzus persicae) and non-vector of PPV (Aphis nerii) aphid species demonstrating acquisition of viral targets by both vector and non-vector aphids. The number of viruliferous aphids detected by real-time RT-PCR and nested RT-PCR in a single closed tube was similar in parallel assays, nevertheless the sensitivity provided by real-time RT-PCR was 100 times higher than nested RT-PCR and 1000 times higher than DASI-ELISA and conventional RT-PCR. The quantities of PPV-RNA targets detected in a single aphid ranged from 40 to more than 2 x 10(3) units. The combined system (immobilization of targets on paper by squash capture and real-time RT-PCR) allows, for the first time, reliable quantitation of PPV targets acquired by individual aphid species and constitute an excellent tool for understanding better PPV epidemiology. (c) 2005 Elsevier B.V. All rights reserved.