Loop-mediated isothermal amplification
This article is missing information about primer design, loop formation.(March 2019) |
Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of
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 the screening of conservative and species-specific nucleotide polymorphisms, in most diagnostics applications a combination of PrimerExplorer and MorphoCatcher is very useful, because it allows for the localization of species-specific nucleotides at 3'-ends of primers to enhance the specificity of reactions.
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
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 – 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
- ^ US patent 6410278, Notomi T, Hase T, "Process for synthesizing nucleic acid", published 2002-06-25, assigned to Eiken Kagaku Kabushiki Kaisha
- PMID 10871386.
- PMID 12144774.
- PMID 31086739.
- PMID 28814081.
- PMID 11708792.
- PMID 15163526.
- PMID 18253475.
- S2CID 19416838.
- S2CID 229449403.
- S2CID 229449403.
- ^ ISBN 978-1-904455-70-7.[page needed]
- ISBN 978-1-903401-20-0. Archived from the original(PDF) on 2018-05-16. Retrieved 2014-05-05.
- PMID 28199317.
- PMID 28945787.
- PMID 21047534.
- PMID 16339303.
- ^ 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 Archived 2016-11-20 at the Wayback Machine
- ^ Ponaka, Reddy V. et al. | AMP 2015 | http://www.ilmar.org.il/diasorin/MBI_AMP2015_MalariaPoster102715.pdf Archived 2016-11-20 at the Wayback Machine
- PMID 17991469.
- ^ Walker, Peter (21 May 2020). "UK coronavirus test with 20-minute wait being trialled". The Guardian.
- PMID 32276051.
- PMID 18524393.
- PMID 24574279.
- PMID 21276085.
- S2CID 209356926.
- S2CID 231869975.
- S2CID 4035223.
- PMID 21679460.
- PMID 18029039.
- PMID 23030060.
- PMID 27984058.
- PMID 33595397.
- PMID 33474990.