Loop-mediated isothermal amplification

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Loop-mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA.[1][2] This may be of use in the future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.

LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler.

Technique[edit]

In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, 4 different primers are used to amplify 6 distinct regions on the target gene, which increases specificity. An additional pair of "loop primers" can further accelerate the reaction.[3] The amount of DNA produced in LAMP is considerably higher than PCR based amplification.

The amplification product can be detected via photometry, measuring the turbidity caused by magnesium pyrophosphate precipitate in solution as a byproduct of amplification.[4] This allows easy visualization by the naked eye or via simple photo metric detection approaches for small volumes. The reaction can be followed in real-time either by measuring the turbidity[5] or by fluorescence using intercalating dyes such as SYTO 9.[6] Dyes such as SYBR green, can be used to create a visible color change that can be seen with the naked eye without the need for expensive equipment, or a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated with the number of copies initially present. Hence, LAMP can also be quantitative. In-tube detection of DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis.[7] Moreover, visual detection of the LAMP amplicons by the unaided eye was based on their ability to hybridize with the complementary gold-bound ss-DNA and thus prevent the normal red to purple-blue color change that would otherwise occur by salt-induced aggregation of the gold particles. So, a LAMP method combined with amplicon detection by AuNP has advantages over previously other methods in terms of reduced assay time, amplicon confirmation by hybridization and use of simpler equipment (i.e., no need for a thermocycler, electrophoresis equipment or a UV trans-illuminator.[8]

Uses and benefits[edit]

LAMP is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages. LAMP has the potential to be used as a simple screening assay in the field or at the point of care by clinicians.[9] Because LAMP is isothermal, which eradicates the need for expensive thermocyclers used in conventional PCR, it may be a particularly useful method for infectious disease diagnosis in low and middle income countries.[10] LAMP is widely being studied for detecting infectious diseases such as tuberculosis,[11] malaria,[12][13][14] and sleeping sickness.[15] In developing regions, it has yet to be extensively validated for other common pathogens.[9]

LAMP has been observed to be less sensitive (more resistant) than PCR to inhibitors in complex samples such as blood, likely due to use of a different DNA polymerase (typically BstBacillus stearothermophilus – DNA polymerase rather than Taq polymerase as in PCR). Several reports describe successful detection of pathogens from minimally processed samples such as heat-treated blood,[16][17] or in presence of clinical sample matrices.[18] This feature of LAMP may be useful in low-resource or field settings where a conventional DNA or RNA extraction prior to diagnostic testing may be impractical.

Limitations[edit]

LAMP is less versatile than PCR, the most familiar nucleic acid amplification technique. LAMP is useful primarily as a diagnostic or detection technique, but is not useful for cloning or myriad other molecular biology applications enabled by PCR. Because LAMP uses 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of the genome, and because primer design is subject to numerous constraints, it is difficult to design primer sets for LAMP "by eye". Free, open-source[19] or commercial software packages are generally used to assist with LAMP primer design, although the primer design constraints mean there is less freedom to choose the target site than with PCR.

In a diagnostic application, this must be balanced against the need to choose an appropriate target (e.g., a conserved site in a highly variable viral genome, or a target that is specific for a particular strain of pathogen). Multiple degenerated sequences may be required to cover the different variant strains of the same species. However, having such a cocktail of primers may lead to non-specific amplification in the late amplification.

Multiplexing approaches for LAMP are less developed than for PCR. The larger number of primers per target in LAMP increases the likelihood of primer-primer interactions for multiplexed target sets. The product of LAMP is a series of concatemers of the target region, giving rise to a characteristic "ladder" or banding pattern on a gel, rather than a single band as with PCR. Although this is not a problem when detecting single targets with LAMP, "traditional" (endpoint) multiplex PCR applications wherein identity of a target is confirmed by size of a band on a gel are not feasible with LAMP. Multiplexing in LAMP has been achieved by choosing a target region with a restriction site, and digesting prior to running on a gel, such that each product gives rise to a distinct size of fragment,[20] although this approach adds complexity to the experimental design and protocol. The use of a strand-displacing DNA polymerase in LAMP also precludes the use of hydrolysis probes, e.g. TaqMan probes, which rely upon the 5'-3' exonuclease activity of Taq polymerase. An alternative real-time multiplexing approach based on fluorescence quenchers has been reported.[21]

Sybrgreen dye may be added to view the real-time LAMP. However, in the late amplification, the primer-dimer amplification may contribute to false positive signal. Unlike traditional SYBR® Green dye–based PCR assays, the melt curve analysis cannot be performed in LAMP to check for the presence of primer-dimers.

References[edit]

  1. ^ Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000). "Loop-mediated isothermal amplification of DNA". Nucleic Acids Res. 28 (12): E63. doi:10.1093/nar/28.12.e63. PMC 102748. PMID 10871386.
  2. ^ US patent 6410278, Notomi T, Hase T, "Process for synthesizing nucleic acid", published 2002-06-25, assigned to Eiken Kagaku Kabushiki Kaisha 
  3. ^ Nagamine K, Hase T, Notomi T (2002). "Accelerated reaction by loop-mediated isothermal amplification using loop primers". Mol. Cell. Probes. 16 (3): 223–9. doi:10.1006/mcpr.2002.0415. PMID 12144774.
  4. ^ Mori Y, Nagamine K, Tomita N, Notomi T (2001). "Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation". Biochem. Biophys. Res. Commun. 289 (1): 150–4. doi:10.1006/bbrc.2001.5921. PMID 11708792.
  5. ^ Mori Y, Kitao M, Tomita N, Notomi T (2004). "Real-time turbidimetry of LAMP reaction for quantifying template DNA". J. Biochem. Biophys. Methods. 59 (2): 145–57. doi:10.1016/j.jbbm.2003.12.005. PMID 15163526.
  6. ^ Njiru ZK, Mikosza AS, Armstrong T, Enyaru JC, Ndung'u JM, Thompson AR (2008). "Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense". PLoS Negl Trop Dis. 2 (1): e147. doi:10.1371/journal.pntd.0000147. PMC 2238707. PMID 18253475. open access
  7. ^ Tomita N, Mori Y, Kanda H, Notomi T (2008). "Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products". Nat Protoc. 3 (5): 877–82. doi:10.1038/nprot.2008.57. PMID 18451795.
  8. ^ Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika (2016). "Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes". PLOS ONE. 11 (3): e0151769. doi:10.1371/journal.pone.0151769. PMC 4803327. PMID 27003504.
  9. ^ a b Sen K, Ashbolt NJ (2011). Environmental microbiology : current technology and water application. Norfolk, UK: Caister Academic Press. ISBN 978-1-904455-70-7.
  10. ^ Macarthur G (2009). Global health diagnostics: research, development and regulation. Academy of Medical Sciences Workshop Report (PDF). Academy of Medical Sciences (Great Britain). ISBN 978-1-903401-20-0.
  11. ^ Geojith G, Dhanasekaran S, Chandran SP, Kenneth J (2011). "Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting". J. Microbiol. Methods. 84 (1): 71–3. doi:10.1016/j.mimet.2010.10.015. PMID 21047534.
  12. ^ Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS (2006). "Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification". Clin. Chem. 52 (2): 303–6. doi:10.1373/clinchem.2005.057901. PMID 16339303.
  13. ^ Ponaka, Reddy V. et al. | ASTMH 2015 | Molecular detection of Plasmodium with Loop Mediated Isothermal Amplification (LAMP) and ensitivity comparison to PET-PCR assay | http://www.ilmar.org.il/diasorin/MBI_MalariaPoster2015-ASTMH_JT_rev3.pdf
  14. ^ Ponaka, Reddy V. et al. | AMP 2015 | http://www.ilmar.org.il/diasorin/MBI_AMP2015_MalariaPoster102715.pdf
  15. ^ Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, Thompson RC, Ndung'u JM (2008). "African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA". Int. J. Parasitol. 38 (5): 589–99. doi:10.1016/j.ijpara.2007.09.006. PMID 17991469.
  16. ^ Curtis KA, Rudolph DL, Owen SM (2008). "Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP)". J. Virol. Methods. 151 (2): 264–70. doi:10.1016/j.jviromet.2008.04.011. PMID 18524393.
  17. ^ Sattabongkot J, Tsuboi T, Han ET, Bantuchai S, Buates S (2014). "Loop-mediated isothermal amplification assay for rapid diagnosis of malaria infections in an area of endemicity in Thailand". J. Clin. Microbiol. 52 (5): 1471–7. doi:10.1128/JCM.03313-13. PMC 3993686. PMID 24574279.
  18. ^ Francois P, Tangomo M, Hibbs J, Bonetti EJ, Boehme CC, Notomi T, Perkins MD, Schrenzel J (2011). "Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications". FEMS Immunol. Med. Microbiol. 62 (1): 41–8. doi:10.1111/j.1574-695X.2011.00785.x. PMID 21276085.
  19. ^ Torres C, Vitalis EA, Baker BR, Gardner SN, Torres MW, Dzenitis JM (2011). "LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures". BMC Bioinformatics. 12: 240. doi:10.1186/1471-2105-12-240. PMC 3213686. PMID 21679460. open access
  20. ^ Iseki H, Alhassan A, Ohta N, Thekisoe OM, Yokoyama N, Inoue N, Nambota A, Yasuda J, Igarashi I (2007). "Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites". J. Microbiol. Methods. 71 (3): 281–7. doi:10.1016/j.mimet.2007.09.019. PMID 18029039.
  21. ^ Tanner NA, Zhang Y, Evans TC (2012). "Simultaneous multiple target detection in real-time loop-mediated isothermal amplification". BioTechniques. 53 (2): 81–9. doi:10.2144/0000113902. PMID 23030060.