Protocol | Published:

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

Nature Protocols volume 3, pages 877882 (2008) | Download Citation

Subjects

This article has been updated

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    et al. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).

  2. 2.

    , , & Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 10, 413–417 (1992).

  3. 3.

    , , & Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 11, 1026–1030 (1993).

  4. 4.

    , , & Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280 (1991).

  5. 5.

    & Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

  6. 6.

    , , , & Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17, 804–807 (1999).

  7. 7.

    , , & Amplification of target-specific, ligation-dependent circular probe. Gene 211, 277–285 (1998).

  8. 8.

    Nucleic acid sequence-based amplification. Nature 350, 91–92 (1991).

  9. 9.

    et al. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87, 1874–1878 (1990).

  10. 10.

    et al. Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20, 1691–1696 (1992).

  11. 11.

    et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000).

  12. 12.

    et al. Detection of Japanese yam mosaic virus by RT-LAMP. Archives of Virology 148, 1713–1720 (2003).

  13. 13.

    , , & Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150–154 (2001).

  14. 14.

    & Indicator for titration of calcium in presence of magnesium using disodium dihydrogen ethylenediamine tetraacetate. Anal. Chem. 28, 882–884 (1956).

  15. 15.

    & Fluorometric determination of calcium in blood serum. Anal. Chem. 35, 1238–1240 (1963).

  16. 16.

    & Fluorescence techniques in the microdetermination of metals in biological materials. Utility of 2, 4-bis-(N,N′-di-(carboxymethyl)aminomethyl) fluorescein in the fluorometric estimation of Al+3, alkaline earths, Co+2, Cu+2, Ni+2, and Zn+2 in micromolar concentrations. Anal. Chem. 35, 1035–1044 (1963).

  17. 17.

    & Metallochromic indicators. IV. A note on the preparation and properties of “Calcein”. Collect. Czech. Chem. Commun. 23, 622–627 (1958).

  18. 18.

    Fluorimetric determination of calcium in serum with calcein: complexation of calcein with calcium and alkali metals. Anal. Chim. Acta. 209, 303–308 (1988).

  19. 19.

    , , , & Loop-mediated isothermal amplification reaction using a nondenatured template. Clin. Chem. 47, 1742–1743 (2001).

Download references

Acknowledgements

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

Author information

Affiliations

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

    • Norihiro Tomita
    • , Yasuyoshi Mori
    • , Hidetoshi Kanda
    •  & Tsugunori Notomi

Authors

  1. Search for Norihiro Tomita in:

  2. Search for Yasuyoshi Mori in:

  3. Search for Hidetoshi Kanda in:

  4. Search for Tsugunori Notomi in:

Corresponding author

Correspondence to Norihiro Tomita.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2008.57

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.