Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII

Grant A Chambers; Andrew D W Geering; Paul Holford; Georgios Vidalakis; Nerida J Donovan

doi:10.1016/j.jviromet.2021.114330

Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII

J Virol Methods. 2022 Jan:299:114330. doi: 10.1016/j.jviromet.2021.114330. Epub 2021 Oct 11.

Authors

Grant A Chambers¹, Andrew D W Geering², Paul Holford³, Georgios Vidalakis⁴, Nerida J Donovan⁵

Affiliations

¹ NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Private Bag 4008, Narellan, NSW, 2567, Australia; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, GPO Box 267, Brisbane, Queensland, 4001, Australia. Electronic address: grant.chambers@dpi.nsw.gov.au.
² Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, GPO Box 267, Brisbane, Queensland, 4001, Australia.
³ Western Sydney University, School of Science, LB 1797, Penrith, NSW, 2752, Australia.
⁴ Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, CA, United States.
⁵ NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Private Bag 4008, Narellan, NSW, 2567, Australia.

PMID: 34648820
DOI: 10.1016/j.jviromet.2021.114330

Abstract

An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.

Keywords: Citrus; Diagnostics; Real-time RT-qPCR; TaqMan®; Viroid.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Australia
Citrus* / virology
Plant Viruses* / isolation & purification
Real-Time Polymerase Chain Reaction* / methods
Sensitivity and Specificity
Viroids* / genetics
Viroids* / isolation & purification