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Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

Nature Protocols volume 3, pages 877–882 (2008)Cite this article

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Abstract

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30–60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.

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Figure 1: Principle of loop-mediated isothermal amplification (LAMP) method.
Figure 2: Principle of detection using a fluorescent metal indicator (calcein).
Figure 3: Detection of the loop-mediated isothermal amplification (LAMP) reaction using fluorescent metal indicator.
Figure 4: Fluorescence spectra of the reaction solution after the amplification reaction (excitation at 480 nm).
Figure 5: Analysis of the loop-mediated isothermal amplification (LAMP) reaction products using agarose gel electrophoresis.

Change history

  • 12 January 2012

    In the version of this article initially published, the legend for Figure 1 contained errors. In the description of panel b, the sentence "The released single strand forms a loop structure at its 3′ end (structure 3)" should have ended with "a loop structure at its 5′ end (structure 4)." In the description of panel c, "Using self-structure as the template..." should have read "Using structure 5 as the template..." The last sentence of the legend ("Structures 9 and 10 are produced from structures 6 and 8, respectively, and more elongated structures (11, 12) are also produced.") has been replaced with the following: "Specifically, intermediate structures 7a and 9a and structures 5a and 10a (in the yellow boxes) are produced from structures 6 and 8, respectively. Structures 9a and 10a then form structures 9 and 10, respectively, whereas the displaced strands 7a and 5a form the dumbbell-like structures 7 and 5, respectively. More elongated structures (11, 12) are also produced." Finally, in panel c of the figure, structure 9 (with downstream structure 11) and structure 10 (with downstream structure 12) were shown in incorrect locations, and additional structures (5a, 7a, 9a, and 10a) were omitted. These errors have been corrected in the HTML and PDF versions of the article. The authors acknowledge S. Subramanian and R.D. Gomez of the University of Maryland, who brought the error to their attention and supplied the corrected version of Figure 1c and legend.

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Acknowledgements

We thank S. Kojiya for help in preparing the manuscript.

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Authors and Affiliations

  1. Eiken Chemical Co., Ltd., Biochemical Research Laboratory, 1381-3 Shimoishigami, Ohtawara, 324-0036, Tochigi, Japan

    Norihiro Tomita, Yasuyoshi Mori, Hidetoshi Kanda & Tsugunori Notomi

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Correspondence to Norihiro Tomita.

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Tomita, N., Mori, Y., Kanda, H. et al. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3, 877–882 (2008). https://doi.org/10.1038/nprot.2008.57

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