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 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.

Technique[edit]

LAMP is an isothermal nucleic acid amplification. 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. Detection of amplification product can be determined via photometry for turbidity caused by an increasing quantity of Magnesium pyrophosphate in solution as a byproduct of amplification[3] or with addition of SYBR green, a color change can be seen with naked eyes without the need for expensive equipments. Also 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.[4] Real-time detection is also possible using intercalating dyes such as SYTO 9.[5]

Uses and benefits[edit]

LAMP has the potential to be used as a simple screening assay in the field or at the point of care by clinicians.[6] As previously mentioned, 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.[7]

LAMP is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages. 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 identify 6 distinct regions on the target gene, which adds highly to the specificity. An additional pair of "loop primers" can further accelerate the reaction.[8] Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The corresponding release of pyrophosphate results in visible turbidity due to precipitation, which allows easy visualization by the naked eye, especially for larger reaction volumes or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity[9] or the signals from DNA produced via fluorescent dyes that intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. While LAMP is widely being studied for detecting infectious diseases such as tuberculosis,[10] malaria,[11] and sleeping sickness.[12] In developing regions, it has yet to be extensively validated for other common pathogens.[6]

LAMP has been observed to be less sensitive than PCR to inhibitors in complex samples such as blood, likely due to use of a different DNA polymerase (typically Bst 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,[13][14] or in presence of clinical sample matrices.[15] 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[16] 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).

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,[17] 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.[18]

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. ^ 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. 
  4. ^ 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. 
  5. ^ 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 publication - free to read
  6. ^ 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. 
  7. ^ Macarthur G (2009). Global health diagnostics: research, development and regulation. Academy of Medical Sciences Workshop Report. Academy of Medical Sciences (Great Britain). ISBN 978-1-903401-20-0. 
  8. ^ 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. 
  9. ^ 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. 
  10. ^ 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. 
  11. ^ 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. 
  12. ^ 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. 
  13. ^ 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. 
  14. ^ 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. PMID 24574279. 
  15. ^ 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. 
  16. ^ 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 publication - free to read
  17. ^ 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. 
  18. ^ 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.