Abstract
High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations. Biotechniques 20:968–970
Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18:62–63
Osman F, Olineka T, Hodzic E, Golino D, Rowhani A (2012) Comparative procedures for sample processing and quantitative PCR detection of grapevine viruses. J Virol Methods 179:303–310
Schrader C, Schielke A, Ellerbroek L, Johne R (2012) PCR inhibitors–occurrence, properties and removal. J Appl Microbiol 113:1014–1026
Roistacher CN (1991) Graft-transmissible diseases of citrus: Handbook for detection and diagnosis. International Organization of Citrus Virologists, Food and Agriculture Organization of the United Nations, Rome
Romero-Durbán J, Cambra M, Duran-Vila N (1995) A simple imprint-hybridization method for detection of viroids. J Virol Methods 55:37–47
Palacio-Bielsa A, Foissac X, Duran-Vila N (1999) Indexing of citrus viroids by imprint hybridisation. Eur J Plant Pathol 105:897–903
Fonseca ME, Marcellino LH, Gander E (1996) A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes. J Virol Methods 57:203–207
Rivera-Bustamante RF, Gin R, Semancik JS (1986) Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system. Anal Biochem 156:91–95
Ito T, Ieki H, Ozaki K (2002) Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. J Virol Methods 106:235–239
Osman F, Dang T, Bodaghi S, Vidalakis G (2017) One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts. J Virol Methods 245:40–52
Gallagher SR (2017) Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr Protoc Immunol 116:A.3L.1–A.3L.14
Saponari M, Manjunath K, Yokomi RK (2008) Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan®). J Virol Methods 147:43–53
Acknowledgments
Support was provided by the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA), Hatch (project 1020106), and the National Clean Plant Network (NCPN) which operates under the auspices of USDA Animal and Plant Health Inspection Service (APHIS) (12-8100-1544-CA; 14-, 15-, 16-8130-0419-CA; AP17PPQS&T00C118; AP18PPQS&T00C107). The findings and conclusions in this publication are those of the author(s) and should not be construed to represent any official USDA or US government determination or policy.
URLs to sponsors’ websites are listed below.
1. CRB: https://www.citrusresearch.org
2. USDA NIFA: https://nifa.usda.gov
3. NCPN: http://nationalcleanplantnetwork.org.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dang, T. et al. (2022). High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G. (eds) Viroids. Methods in Molecular Biology, vol 2316. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1464-8_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1464-8_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1463-1
Online ISBN: 978-1-0716-1464-8
eBook Packages: Springer Protocols