Biology:Loop-mediated isothermal amplification

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Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA[1] and a low-cost alternative to detect certain diseases that was invented in 2000 at the University of Tokyo.[2] Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP 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

In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C (140-149 °F) 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[citation needed] than PCR-based amplification. Primer design could be performed using several programs, such as PrimerExplorer, MorphoCatcher,[4] and NEB LAMP Primer Design Tool. For screening of conservative and species-specific nucleotide polymorphisms in the most of diagnostics applications a combination of PrimerExplorer and MorphoCatcher is very useful, because allows to localize the species-specific nucleotides at 3'-ends of primers for enhancing the specificity of reaction.

Schema of a LAMP of nucleic acid biomarkers from raw untreated wastewater samples, to rapidly quantify human-specific mitochondrial DNA (mtDNA).[5]

The amplification product can be detected via photometry, measuring the turbidity caused by magnesium pyrophosphate precipitate in solution as a byproduct of amplification.[6] This allows easy visualization by the naked eye or via simple photometric detection approaches for small volumes. The reaction can be followed in real-time either by measuring the turbidity[7] or by fluorescence using intercalating dyes such as SYTO 9.[8] 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 for 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 LAMP DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis.[9]

Another method for visual detection of the LAMP amplicons by the unaided eye was based on their ability to hybridize with complementary gold nanoparticle-bound (AuNP) single-stranded DNA (ssDNA) and thus prevent the normal red to purple-blue color change that would otherwise occur during salt-induced aggregation of the gold particles. So, a LAMP method combined with amplicon detection by AuNP can have advantages over 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).[10][11]

Uses and benefits

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.[12] 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.[13] LAMP is widely being studied for detecting infectious diseases such as filariasis,[14] Zika Virus,[15] tuberculosis,[16] malaria,[17][18][19] sleeping sickness,[20] and SARS-CoV-2.[21][22] In developing regions, it has yet to be extensively validated for other common pathogens.[12]

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 Bst – Bacillus 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,[23][24] or in presence of clinical sample matrices.[25] 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.

LAMP has also been used in helping identify body fluids. With its simplicity, researchers are able to test one or more samples with little hands on time which is helping cut down the time needed to get results. Researchers have also been able to add factors to make identification even more simple including metal-indicator dye and phenol red to be able to use a smartphone and the naked eye respectively to analyze the results.[26][27][28]

Limitations

LAMP is less versatile than PCR, the most well-established nucleic acid amplification technique. LAMP is useful primarily as a diagnostic or detection technique, but is not useful for cloning or many 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[29] 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. A consequence of having such a cocktail of primers can be 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,[30] 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.[31]

SYBR green dye may be added to view LAMP in real-time. However, in the late amplification, primer-dimer amplification may contribute to a false positive signal. The use of inorganic pyrophosphatase in a SYBR reaction mix allows the use of melt analysis to distinguish correct amplification [32]

Although different mitigation strategies have been proposed for false-positive results in assays based on this method, nonspecific amplification due to various factors including the absence of temperature gating mechanisms is one of the major limitations of Loop-mediated isothermal amplification.[33][34]

Lastly, because LAMP requires maintained, elevated incubation temperatures (60–65 °C), some sort of heating mechanism, thermostat, and/or insulator is required (though not necessarily a thermal cycler). This requirement makes LAMP less ideally suited for in the field, point-of-care diagnostics which would ideally function at ambient temperature.

See also

References

  1. ↑ Notomi T, Hase T, "Process for synthesizing nucleic acid", US patent 6410278, published 2002-06-25, assigned to Eiken Kagaku Kabushiki Kaisha
  2. ↑ "Loop-mediated isothermal amplification of DNA". Nucleic Acids Res. 28 (12): 63e–63. 2000. doi:10.1093/nar/28.12.e63. PMID 10871386. 
  3. ↑ "Accelerated reaction by loop-mediated isothermal amplification using loop primers". Mol. Cell. Probes 16 (3): 223–9. 2002. doi:10.1006/mcpr.2002.0415. PMID 12144774. 
  4. ↑ Shirshikov, Fedor V.; Pekov, Yuri A.; Miroshnikov, Konstantin A. (2019-04-26). "MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method" (in en). PeerJ 7: e6801. doi:10.7717/peerj.6801. ISSN 2167-8359. PMID 31086739. 
  5. ↑ Yang, Zhugen; Xu, Gaolian; Reboud, Julien; Kasprzyk-Hordern, Barbara; Cooper, Jonathan M. (19 September 2017). "Monitoring Genetic Population Biomarkers for Wastewater-Based Epidemiology". Analytical Chemistry 89 (18): 9941–9945. doi:10.1021/acs.analchem.7b02257. PMID 28814081. 
  6. ↑ "Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation". Biochem. Biophys. Res. Commun. 289 (1): 150–4. 2001. doi:10.1006/bbrc.2001.5921. PMID 11708792. 
  7. ↑ "Real-time turbidimetry of LAMP reaction for quantifying template DNA". J. Biochem. Biophys. Methods 59 (2): 145–57. 2004. doi:10.1016/j.jbbm.2003.12.005. PMID 15163526. 
  8. ↑ "Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense". PLOS Negl Trop Dis 2 (1): e147. 2008. doi:10.1371/journal.pntd.0000147. PMID 18253475. 
  9. ↑ "Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products". Nat Protoc 3 (5): 877–82. 2008. doi:10.1038/nprot.2008.57. PMID 18451795. 
  10. ↑ Arunrut, Narong; Jitrakorn, Sarocha; Saksmerprome, Vanvimon; Kiatpathomchai, Wansika (August 2019). "Double-Loop-Mediated Isothermal Amplification (D-LAMP) using colourimetric gold nanoparticle probe for rapid detection of infectious Penaeus stylirostris densovirus (PstDNV) with reduced false-positive results from endogenous viral elements". Aquaculture 510: 131–137. doi:10.1016/j.aquaculture.2019.05.049. 
  11. ↑ Arunrut, Narong; Tondee, Benyatip; Khumwan, Pakapreud; Kampeera, Jantana; Kiatpathomchai, Wansika (February 2021). "Rapid and sensitive colorimetric detection of microsporidian Enterocytozoon hepatopenaei (EHP) based on spore wall protein (SWP) gene using loop-mediated isothermal amplification combined with DNA functionalized gold nanoparticles as probes". Aquaculture 533: 736206. doi:10.1016/j.aquaculture.2020.736206. 
  12. ↑ 12.0 12.1 Environmental microbiology : current technology and water application. Norfolk, UK: Caister Academic Press. 2011. ISBN 978-1-904455-70-7. [page needed]
  13. ↑ 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. http://tbevidence.org/documents/rescentre/books/globalhealthdx.pdf. Retrieved 2014-05-05. 
  14. ↑ Poole, Catherine B.; Li, Zhiru; Alhassan, Andy; Guelig, Dylan; Diesburg, Steven; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C. et al. (2017). "Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP)". PLOS ONE 12 (2): e0169011. doi:10.1371/journal.pone.0169011. ISSN 1932-6203. PMID 28199317. Bibcode: 2017PLoSO..1269011P. 
  15. ↑ Calvert, Amanda E.; Biggerstaff, Brad J.; Tanner, Nathan A.; Lauterbach, Molly; Lanciotti, Robert S. (2017). "Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP)". PLOS ONE 12 (9): e0185340. doi:10.1371/journal.pone.0185340. ISSN 1932-6203. PMID 28945787. Bibcode: 2017PLoSO..1285340C. 
  16. ↑ "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. 2011. doi:10.1016/j.mimet.2010.10.015. PMID 21047534. 
  17. ↑ "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. 2006. doi:10.1373/clinchem.2005.057901. PMID 16339303. 
  18. ↑ Ponaka, Reddy V. et al. | ASTMH 2015 | Molecular detection of Plasmodium with Loop Mediated Isothermal Amplification (LAMP) and sensitivity comparison to PET-PCR assay | http://www.ilmar.org.il/diasorin/MBI_MalariaPoster2015-ASTMH_JT_rev3.pdf
  19. ↑ Ponaka, Reddy V. et al. | AMP 2015 | http://www.ilmar.org.il/diasorin/MBI_AMP2015_MalariaPoster102715.pdf
  20. ↑ "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. 2008. doi:10.1016/j.ijpara.2007.09.006. PMID 17991469. 
  21. ↑ Walker, Peter (21 May 2020). "UK coronavirus test with 20-minute wait being trialled". The Guardian. https://www.theguardian.com/world/2020/may/21/uk-coronavirus-test-with-20-minute-wait-being-trialled. 
  22. ↑ Park, Gun-Soo; Ku, Keunbon; Baek, Seung-Hwa; Kim, Seong-Jun; Kim, Seung Il; Kim, Bum-Tae; Maeng, Jin-Soo (2020). "Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)". The Journal of Molecular Diagnostics 22 (6): 729–735. doi:10.1016/j.jmoldx.2020.03.006. PMID 32276051. 
  23. ↑ "Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP)". J. Virol. Methods 151 (2): 264–70. 2008. doi:10.1016/j.jviromet.2008.04.011. PMID 18524393. 
  24. ↑ "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. 2014. doi:10.1128/JCM.03313-13. PMID 24574279. 
  25. ↑ "Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications". FEMS Immunol. Med. Microbiol. 62 (1): 41–8. 2011. doi:10.1111/j.1574-695X.2011.00785.x. PMID 21276085. 
  26. ↑ Jackson, Kimberly R.; Layne, Tiffany; Dent, David A.; Tsuei, Anchi; Li, Jingyi; Haverstick, Doris M.; Landers, James P. (March 2020). "A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection". Forensic Science International: Genetics 45: 102195. doi:10.1016/j.fsigen.2019.102195. ISSN 1872-4973. PMID 31835180. 
  27. ↑ Layne, Tiffany; Jackson, Kimberly; Scott, Anchi; Tanner, Nathan A.; Piland, Annie; Haverstick, Doris M.; Landers, James P. (2021). "Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification" (in en). Journal of Forensic Sciences 66 (3): 1033–1041. doi:10.1111/1556-4029.14682. ISSN 1556-4029. PMID 33559876. https://onlinelibrary.wiley.com/doi/abs/10.1111/1556-4029.14682. 
  28. ↑ Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi (2018-07-01). "Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay" (in en). International Journal of Legal Medicine 132 (4): 975–981. doi:10.1007/s00414-017-1661-z. ISSN 1437-1596. PMID 28803416. https://doi.org/10.1007/s00414-017-1661-z. 
  29. ↑ "LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures". BMC Bioinformatics 12: 240. 2011. doi:10.1186/1471-2105-12-240. PMID 21679460. 
  30. ↑ Iseki, Hiroshi; Alhassan, Andy; Ohta, Naomi; Thekisoe, Oriel M.M.; Yokoyama, Naoaki; Inoue, Noboru; Nambota, Andrew; Yasuda, Jun et al. (December 2007). "Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites". Journal of Microbiological Methods 71 (3): 281–7. doi:10.1016/j.mimet.2007.09.019. PMID 18029039. 
  31. ↑ "Simultaneous multiple target detection in real-time loop-mediated isothermal amplification". BioTechniques 53 (2): 81–9. August 2012. doi:10.2144/0000113902. PMID 23030060. 
  32. ↑ "Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values". J Microbial Methods 132: 41–45. January 2017. doi:10.1016/j.mimet.2016.10.020. PMID 27984058. 
  33. ↑ Habibzadeh, Parham; Mofatteh, Mohammad; Silawi, Mohammad; Faghihi, Mohammad Ali; Ghavami, Saeid (2021). "Molecular diagnostic assays for COVID-19: an overview". Critical Reviews in Clinical Laboratory Sciences 58 (6): 385–398. doi:10.1080/10408363.2021.1884640. PMID 33595397. 
  34. ↑ H Moehling, Taylor J; Choi, Gihoon; Dugan, Lawrence; Salit, Marc; Meagher, Robert (2021). "LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community". Expert Review of Molecular Diagnostics 21 (1): 43–61. doi:10.1080/14737159.2021.1873769. PMID 33474990. 



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