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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZING, DETECTING, AND ELIMINATING PATHOGENS TO ENABLE THE SAFE INTRODUCTION OF PLANT GENETIC RESOURCES

Location: National Germplasm Resources

Title: Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

Authors
item Komorowska, Beata -
item Fiore, Nicola -
item Zamorano, Alan -
item Li, Ruhui

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 12, 2014
Publication Date: March 24, 2014
Citation: Komorowska, B., Fiore, N., Zamorano, A., Li, R. 2014. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis. Archives of Virology. Molecular and Cellular Probes 28:186-191.

Interpretive Summary: Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) causes disease in certain cherry varieties. The current method to detect CGRMV and CNRMV includes molecular techniques which either detect them individually or do not differentiate them. In this study, we have developed a molecular technique to detect and differentiate the two viruses in a single reaction, thereby saving time and reagents. The assay is also simple, fast and reliable. This will be useful to scientists for the routine diagnosis of these cherry viruses.

Technical Abstract: In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a pair of virus-specific primers for each of them or multiplex reaction that included one specific primer set for each virus. These two strategies allowed us to confirmed virus infection in all tested samples. In 17 field samples the technique revealed samples positive for CNRMV or CGRMV as well as positive for both viruses. In addition, HRM analysis made it possible to differentiate clearly between CNRMV and CGRMV by HRM curves. Moreover, sequence variations among CNRMV and CGRMV isolates have been observed from the HRM peaks, as confirmed by sequencing. The HRM real-time PCR provides sensitive, automated and rapid tool to detect and differentiate between CNRMV and CGRMV isolates.

Last Modified: 11/26/2014
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