One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts

•Developed RT-qPCR for species-specific multiplex detection of three citrus viroids.•RT-qPCR detected viroids in citrus and non-citrus hosts from around the world.•RT-qPCR co-detected and identified targeted viroids in single and mixed infections.•RT-qPCR differentiated viroid species in SYBR Green...

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Bibliographic Details
Published in:Journal of virological methods 2017-07, Vol.245, p.40-52
Main Authors: Osman, Fatima, Dang, Tyler, Bodaghi, Sohrab, Vidalakis, Georgios
Format: Article
Language:English
Subjects:
Citrus - virology
Citrus Nursery Stock Pest Cleanliness Program
Diagnostics
DNA Primers
Germplasm
Graft-transmissible pathogens
Lycopersicon esculentum - virology
Multiplex Polymerase Chain Reaction - methods
National Clean Plant Network
Organic Chemicals
Plant Diseases - virology
Propagative materials
Viroids - genetics
Viroids - isolation & purification
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Summary:•Developed RT-qPCR for species-specific multiplex detection of three citrus viroids.•RT-qPCR detected viroids in citrus and non-citrus hosts from around the world.•RT-qPCR co-detected and identified targeted viroids in single and mixed infections.•RT-qPCR differentiated viroid species in SYBR Green I positive samples. A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2017.03.007