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Reliable and inexpensive Real-time RT-PCR method for Apple stem grooving virus and Apple stem pitting virus detection

Eunice Beaver-Kanuya: Washington State University

<div><em>Apple stem grooving virus</em> (ASGV) and <em>Apple stem pitting virus</em> (ASPV) negatively impact production, maintenance, and distribution of apples and other <em>Malus</em> species world-wide. Control of these two pathogens relies on rapid detection and diagnosis, particularly to identify newly infected plants for removal before they become a source of inoculum for further spread. Despite the increasing diversity of isolates found by high-throughput sequencing, little effort has been made to reassess or improve the diagnostic assays used for these two viruses. In this study we aimed to develop robust and reliable real-time PCR assays to meet these requirements. Primers and probes were designed against alignments of representative extant sequences from across the globe, and reaction conditions were optimized for speed, sensitivity, and specificity. Each assay was validated against a panel of ASGV and ASPV isolates and against related non-target virus species and common host species to ensure reaction specificity. These new assays showed increased detection of these two viruses in a quarantine setting. Similar results were obtained comparing the real-time RT-PCR assay and the high-throughput sequencing method in a panel of 13 samples. Adoption of the real-time RT-PCR platform for these two viruses reduced time-to-answer from 2-3 hours to 1:20 hour. The new method worked well for both domestic and foreign apple isolates and reduced the time and the cost for diagnosis.</div>