A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

G. A. Chambers; A. D. W. Geering; Paul Holford; M. A. Kehoe; G. Vidalakis; N. J. Donovan

doi:10.1007/s13313-023-00903-1

A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

G. A. Chambers, A. D. W. Geering, Paul Holford, M. A. Kehoe, G. Vidalakis, N. J. Donovan

Western Sydney University

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.

Original language	English
Pages (from-to)	121-132
Number of pages	12
Journal	Australasian Plant Pathology
Volume	52
Issue number	2
DOIs	https://doi.org/10.1007/s13313-023-00903-1
Publication status	Published - Mar 2023

Bibliographical note

Publisher Copyright:
© 2023, The Author(s).

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Keywords

RT-LAMP
Graft-transmissible
Scalybutt
Plant pathogen detection
Scaly bark
CEVd

UN SDGs

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10.1007/s13313-023-00903-1

Cite this

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title = "A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus",

abstract = "Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.",

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author = "Chambers, {G. A.} and Geering, {A. D. W.} and Paul Holford and Kehoe, {M. A.} and G. Vidalakis and Donovan, {N. J.}",

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doi = "10.1007/s13313-023-00903-1",

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AU - Holford, Paul

AU - Kehoe, M. A.

AU - Vidalakis, G.

AU - Donovan, N. J.

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N2 - Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.

AB - Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.

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A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

Abstract

Bibliographical note

Open Access - Access Right Statement

Keywords

UN SDGs

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