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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research Unit » Research » Publications at this Location » Publication #269922

Title: Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

Author
item QUITO-AVILA, DIEGO - Oregon State University
item Martin, Robert

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2011
Publication Date: 1/15/2012
Citation: Quito-Avila, D., Martin, R.R. 2012. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections. Journal of Virological Methods. 179(1):38-44.

Interpretive Summary: Quantitative polymerase chain reaction (qPCR) assays have been developed for the detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV). RBDV is one of the most common viruses of raspberry and blackberry worldwide, and RLMV is very common in North America and Europe but has not been studied in other parts of the world. The primers were developed based on conserved regions of the polymerase gene using sequences from GenBank and corresponding sequences of all isolates used in this study. The qPCR assays developed were able to detect as few as 30, or 300 copies of the RBDV and RLMV genomes, respectively. Using these assays, it was shown that the titer of RBDV in plants increases approximately 400-fold when in mixed infections with RLMV compared to RBDV in single infections in the same cultivar. The titer of RLMV was not influenced by the presence of RBDV. In addition, with the qPCR assay it was possible to detect RLMV in single aphids. These assays are being used to study aphid transmission of RLMV and to better understand the interaction between the two viruses in causing crumbly fruit and decline in raspberry.

Technical Abstract: Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conserved fragments of the polymerase region of each virus and were effective for the detection of different isolates tested in this study. The RBDV assay amplified a 94 bp amplicon and was able to detect as few as 30 viral copies. Whereas the RLMV assay amplified a 180 bp amplicon and detected as few as 300 viral copies from plant and aphid RNA extracts. A comparison with conventional detection methods revealed that that both assays were significantly more sensitive than RT-PCR. The sensitivity of the RLMV assay was also tested on single aphids after a fixed acquisition access period (AAP). In addition, the 24 assays revealed a novel synergistic interaction between the two viruses, where the concentration of RBDV was enhanced ~400-fold when it occurred in combination with RLMV compared to its concentration in single infections. The significance of this finding and the importance of the development of real-time RT-PCR assays for the detection of RBDV and RLMV are discussed.