A rapid isothermal assay for the detection of Hop stunt viroid in hop plants (Humulus lupulus), and its application in disease surveys

• Published in: Journal of virological methods 2017-07, Vol.245, p.81-85
• Main Authors: Kappagantu, Madhu, Villamor, Dan Edward V., Bullock, Jeff M., Eastwell, Kenneth C.
• Format: Article
• Language: English
• Subjects: Citrus - virology; DNA, Viral; FTA sample cards; Genome, Viral; Hop stunt viroid; Humulus - virology; Humulus lupulus; Isothermal amplification; Phylogeny; Plant Diseases - virology; Real-Time Polymerase Chain Reaction - methods; Reverse transcription-recombinase polymerase amplification assay; Temperature; Viroids - genetics; Viroids - isolation & purification
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2017.04.002

•A reverse transcription recombinase polymerase amplification assay was developed for rapid and simplified detection of Hop stunt viroid.•Reverse transcription recombinase polymerase amplification assay is compatible with a survey conducted with fiber sample collection cards.•The resulting assay was validated across representative samples of all major variants of Hop stunt viroid and their hosts. Hop stunt disease caused by Hop stunt viroid (HSVd) is a growing threat to hop cultivation globally. HSVd spreads mainly by use of contaminated planting material and by mechanical means. Thorough testing of hop yards and removal of infected bines are critical components of efforts to control the spread of the disease. Reverse transcription-polymerase chain reaction (RT-PCR) has become the primary technique used for HSVd detection; however, sample handling and analysis are technically challenging. In this study, a robust reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed to facilitate analysis of multiple samples. The assay was optimized with all major variants of HSVd from other host species in addition to hop variants. Used in conjunction with sample collection cards, RT-RPA accommodates large sample numbers. Greenhouse and farm samples tested with RT-RPA were also tested with RT-PCR and a 100% correlation between the two techniques was found.