Jun 15, 2020
RPA-based tobacco rattle virus testing method announced by Agdia

Agdia has announced the commercialization of a rapid, PCR-alternative testing to detect tobacco rattle virus (TRV).

Tobacco rattle virus, or tobravirus, affects 400 species of vegetable plants — potatoes included — ornamentals and weeds.

In potatoes, it leads to corky ringspot in tubers, making them undesirable to chip makers and retailers. Further, exterior skin symptoms can be mistaken for potato virus Y, while tuber necrosis symptoms are similar to mop-top virus, making testing necessary to be sure which disease is present.

Symptoms of TRV vary according to plant variety and environmental conditions.

Agdia’s new detection technology, AmplifyRP XRT assay, is based on recombinase polymerase amplification (RPA). This technology promotes the rapid amplification and detection of nucleic acid targets, DNA or RNA, while maintaining a single operating temperature of 39-42C. The AmplifyRP XRT products achieve target sensitivity and specificity comparable to PCR, while having clear advantages over the lab-based technology.

AmplifyRP XRT products do not require a nucleic acid purification step; crude sample extracts are prepared using a simple extraction buffer and tested directly, which makes the testing process simple and saves time. When paired with an isothermal fluorometer, the products make for a quick, easy-to-easy tool that can be implemented in the field or a laboratory, even with those who have limited molecular diagnostic experience.

If a fluorometer isn’t available, samples can be sent to an Agdia Testing Service lab and tested at less cost than PCR testing, said Deborah Groth-Helms, Agdia’s director of quality manager.

Agdia’s RPA advancements for tobacco rattle virus detection were part of phase I of a USDA-NIFA Small Business Innovation Research program grant. The company plans to apply for phase II funding next year, which would be to develop a smaller, more cost-effective fluorometer.

Agdia states their new assay was tested against an extensive collection of confirmed TRV isolates from numerous ornamental hosts, potato and additional vegetable hosts, providing 100% accuracy for detection of true positives. Moreover, no cross-reactivity was observed with a comprehensive panel of pathogens, including barley stripe mosaic virus, beet soilborne virus, cucumber green mottle mosaic virus, kyuri green mottle mosaic virus, Odontoglossum ringspot virus, pea early browning virus, pepper ringspot virus, potato mop-top virus, ribgrass mosaic virus, soilborne wheat mosaic virus, tobacco mild green mosaic virus, tobacco mosaic virus-c and tomato mosaic virus.

Sensitivity for this assay is comparable to that observed with the published conventional and qPCR to which it was compared. This product includes an endogenous RNA control and was developed to test leaf and sprout tissue. Moreover, Agdia validated this product for testing tuber tissue directly. For more information, visit www.agdia.com.

Agdia acknowledged assistance in validating the testing from the labs of Stewart Gray and Hanu Pappu.

Other plants affected

TRV infects several species of herbaceous perennials, causing potentially severe disease in some species. Ornamental hosts include Astilbe, Dicentra, Epimedium, Gladiolus, Heuchera, Hosta, Hyacinthus, Paeonia, Narcissus, Tagetes, Tulipa and Vinca.

Symptomology varies according to host, and many are latently infected, with the virus proliferating in the root system. Nevertheless, TRV infections can express as angular, yellow-white line patterns and ringspots on the foliage of Paeonia spp. and mottling, mosaic and necrosis on Hosta foliage. Foliar symptoms can reduce the aesthetic value of ornamentals; however, it is unclear how TRV infections affect the overall vigor of asymptomatic hosts.

Additional economically important host genera include allium, several in brassicaceae, capsicum, cucumis and phaseolus.






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