Abstract
Loop-mediated isothermal amplification (LAMP) is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activity of Bacillus subtilis-derived (Bst) DNA polymerase. The end result of strand displacement and loop formation and synthesis is the single-temperature amplification of a highly specific fragment from a DNA template at a much greater titre than that obtained with polymerase chain reaction. With LAMP, there are several methods to determine a positive reaction. Presented here are three alternative methods: gel electrophoresis, hydroxynaphthol blue colorimetric dye, and the fluorescent intercalating PicoGreen® reagent.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63.
Nagamine, K., Hase, T. and Notomi, T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. and Cell. Probes 16, 223–229.
Fukuta, S., Mizikami, Y., Ishida, A., Ueda, J., Kanabe, M. and Ishimoto,Y. (2003) detection of Japanese yam mosaic virus by RT-LAMP. Arch. Virol. 148, 1713–1720.
Nie, X. (2005) Reverse Transcription Loop-mediated isothermal amplification of DNA for Detection of Potato virus Y. Plant Dis. 89, 605–610.
Savan, R., Igarashi, A., Matsuoka, S. and Sakai, M. (2004) Sensitive and rapid detection of Edwardsiellosis in fish by a loop-mediated isothermal amplification method. Appl. Environ. Microbiol. 70, 621–624.
Dukes, J. P., King, D. P. and Alexandersen, S. (2006) Novel reverse-transcription loop-mediated isothermal amplification for rapid detection. Arch. Virol. 151, 1093–1106.
Varga, A. and James, D. (2006) Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus. J. Virol. Methods 138, 184–190.
Tomlinson, J., Barker, I. and Boonham, N. (2007) Faster, simpler, more specific methods for improved molecular detection of Phytophthora ramorum in the field. Appl. Environ. Microbiol. 73, 4040–4047.
Tomlinson, J. and Boonham, N. (2008) Potential of LAMP for detection of plant pathogens. Common W. Agric. Bur. Rev. Perspect. Agric Vet. Sci. Nutr. Nat. Resour. 3, 1–7.
Fukuta, S., Ohishi, K., Yoshida, K., Mizikami, Y., Ishida, A. and Kanabe, M. (2004) Development of immunocapture reverse-transcription loop mediated isolthermal amplification for the detection of Tomato spotted wilt virus from chrysanthemum. J. Virol. Methods 121, 49–55.
Goto, M., Honda, E., Ogura, A., Nomoto, A. and Hanaki, K. (2009) Colormetric detection of loop mediated isothermal amplification reaction by using hydroxynapthol blue. Biotechnqiues 46, 167–172.
Iwamoto, T., Sonobe, T. and Hayashi, K. (2003) Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 41, 2616–2622.
Mori, Y., Nagamine, K., Tomita, N. and Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150–154.
Mori, Y., Hirano, T. and Notomi, T. (2006) Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol. 6, 3.
Tomlinson, J. A., Dickinson, M. J. and Boonham, N. (2010) Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. Phytopathology 100, 143–149.
Harper, S. J., Ward, L. I. and Clover, G. R. G. (2010) Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications. Phytopathology 84, 456–461.
En, F. X., Wei, X., Jian, L. and Qin, C. (2008) Loop-mediated isothermal amplification establishment for detection of pseudorabies virus. J. Virol. Methods 151, 35–39.
Kubota, R., Vine, B. G., Alvarez, A. M. and Jenkins, D. M. (2008) Detection of Ralstonia solanacaerum by loop-mediated isothermal amplification. Phytopathology 98, 1045–1051.
Okuda, M., Matsumoto, M., Takana, Y., Subandiah, S. and Iwanami, T. (2005) Characterisation of the tufB-secE-nusG-rplKAJL-rpoB gene cluster of the citrus greening organism and detection by loop-mediated isothermal amplification. Plant Dis. 89, 705–711.
Song, T., Toma, C., Nakasone, N., Iwanaga, M. (2005) Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop mediated isothermal amplification method. FEMS Microbiol. Lett. 243, 259–263.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ward, L.I., Harper, S.J. (2012). Loop-Mediated Isothermal Amplification for the Detection of Plant Pathogens. In: Sucher, N., Hennell, J., Carles, M. (eds) Plant DNA Fingerprinting and Barcoding. Methods in Molecular Biology, vol 862. Humana Press. https://doi.org/10.1007/978-1-61779-609-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-609-8_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-608-1
Online ISBN: 978-1-61779-609-8
eBook Packages: Springer Protocols