Abstract
Conventionally, diagnostics and phylogenetics of phytoplasmas have been primarily based on the 16S rRNA gene, for which “universal” primers are available that amplify from most phytoplasma 16Sr groups. However, there has been a drive in recent years to develop “universal” primers for other genes that can be used to complement the use of the 16S rRNA gene. This chapter details the use of primers based on the phytoplasma secA gene and describes how these primers can be used in both a single or nested PCR approach for amplification. It also notes the use of appropriate controls that should be undertaken and provides a source for phytoplasma secA sequences that are available in databases that can be used for phylogenetic analyses.
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 subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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
Martini M et al (2007) Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas. Int J Syst Evol Microbiol 57:2037–2051
Martini M, Lee IM (2012) PCR and RFLP analyses based on the ribosomal protein operon. In: Dickinson M, Hodgetts J (eds) Methods in molecular biology. Springer, New York
Kakizawa S et al (2001) Cloning and expression analysis of Phytoplasma protein translocation genes. Mol Plant Microbe Interact 14:1043–1050
Hodgetts J et al (2008) Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of ‘Candidatus Phytoplasma’. Int J Syst Evol Microbiol 58:1826–1837
Bekele B et al (2011) Use of a real-time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathol 60:345–355
Tomlinson JA et al (2005) On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field. Appl Environ Microbiol 71:6702–6710
Cullen DW, Hirsch PR (1998) Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Soil Biol Biochem 30:983–993
Tamura K et al (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbour Press, New York
Wei W et al (2007) Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int J Syst Evol Microbiol 57:1855–1867
Acknowledgement
We would like to thank Defra, BBSRC, DfiD, and the Leverhulme Society for funding various aspects of this work, and Dr Nigel Harrison, University of Florida, Fort Lauderdale, USA for providing us with the secA sequence of the 16SrIV lethal yellows phytoplasma that enabled us to design the initial primers. 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 assays.
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
Dickinson, M., Hodgetts, J. (2013). PCR Analysis of Phytoplasmas Based on the secA Gene. 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_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-089-2_17
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