Abstract
Conventionally, detection of phytoplasmas has been performed by PCR of the 16S rRNA gene, followed by either RFLP or DNA sequencing to determine the phytoplasma 16Sr group. This chapter demonstrates the technique of terminal restriction fragment length polymorphism (T-RFLP), a fingerprinting technique which combines both detection and identification in a single method, with the added benefit of inbuilt controls which removes the risk of false negative results and in addition highlights potential false positive results.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Smart CD et al (1996) Phytoplasma-specific PCR primers based on sequences of the 16S–23S rRNA spacer region. Appl Environ Microbiol 62:2988–2993
Duduk B, Paltrinieri S, Lee IM, Bertaccini A (2012) Nested PCR and RFLP analysis based on the 16S rRNA gene. In: Dickinson M, Hodgetts J (eds) Methods in molecular biology. Springer, New York
Harrison NA, Womack M, Carpio ML (2002) Detection and characterization of a lethal yellowing (16SrIV) group phytoplasma in Canary Island date palms affected by lethal decline in Texas. Plant Dis 86:676–681
Anthony RM, Brown TJ, French GL (2000) Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridisation to an oligonucleotide array. J Clin Microbiol 38:781–788
Liu W-T et al (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522
Hodgetts J et al (2007) Use of terminal restriction fragment length polymorphism (T-RFLP) for identification of phytoplasmas in plants. Plant Pathol 56:357–365
Acknowledgement
This work was completed as part of a Defra Plant Health Fellowship for JH. We would also like to thank all individuals and organizations that have provided us with phytoplasma DNA/infected plant samples to enable us to validate the assay.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hodgetts, J., Dickinson, M. (2013). T-RFLP for Detection and Identification of Phytoplasmas in Plants. In: Dickinson, M., Hodgetts, J. (eds) Phytoplasma. Methods in Molecular Biology, vol 938. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-089-2_20
Download citation
DOI: https://doi.org/10.1007/978-1-62703-089-2_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-088-5
Online ISBN: 978-1-62703-089-2
eBook Packages: Springer Protocols