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Loop-Mediated Isothermal Amplification (LAMP)
LAMP detection of BRCA1 in HeLa cells. 500 ng (Green), 50 ng (Blue), 5 ng (Orange), and NTC (Black) of gDNA in HeLa cells was used in LAMP reaction with LAMP Green™ fluorescent dye using ABI 7500 qPCR machine.
Table of Contents
LAMP vs. PCR
| Basis Of Differentiation | LAMP | PCR |
| Temperature requirements | Is an isothermal process, reaction occurs over a single consistent temperature of 60°C to 65°C throughout | Requires numerous cycles of heating and cooling - reaction occurs over varying temperatures |
| Number of primers used | Uses 4 to 6 primers to recognize 6 to 8 distinct regions | Uses two primers to recognize 2 regions |
| Reaction time | Rapid reaction, takes less than thirty minutes | Slow reactions, typically takes over one hour |
| Sensitivity to sample matrix inhibitors | More tolerant towards sample matrix inhibitors | Highly sensitive to sample matrix inhibitors |
| Visual detection | Amenable to visual detection based on certain factors such as turbidity among others | Not amenable to visual detection |
| Typical yield | Approx. 10 mg-20 mg | Approx. 0.2 mg |
| Equipment needed | Equipment is simpler | Equipment is more complex to accommodate the recurring heating and cooling cycles |
LAMP techniques work very similar to PCR, but can be completed much faster, in roughly 90 minutes. Some variations of LAMP even have shorter test times, ranging from 30-60 min. LAMP techniques have the ability to amplify just a few copies of DNA to 109 copies, and do not require the use of a thermocycler like PCR. LAMP methods are performed under isothermal conditions at a constant temperature range between 60-65°C. The precision of this technique, coupled with its simplicity and amplification efficiency, make it a suitable, portable field test, even in the most remote settings. LAMP carries the added benefit of efficiently amplifying RNA sequences when used in conjunction with reverse transcription techniques. Additionally, various LAMP master mixes are readily, commercially available.
General Protocol & Methodology
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Principle of Loop-Mediated Isothermal Amplification (LAMP), from base target sequence to generation of quantifiable loop complexes produced by targeted primers and polymerization steps. Illustration made using Biorender.
General LAMP techniques employ the use of bacillus stearothermophilus (Bst), a DNA polymerase, which possesses a high strand-displacement activity. In most LAMP methods 6 different primers are designed to recognize 8 distinct regions on a target gene, and amplification will occur if the primers bind and form the intended product.
Two sets of inner primers are used (forward and backward inner primers) along with two sets of outer primers (forward and backward outer primers). The LAMP reaction is initiated by an inner primer containing sequences of sense and antisense strands of the target DNA. Using the stem-loop areas as a stage, DNA polymerase-mediated strand displacement synthesis repeats the elongation events continually. After, denaturation releases single-stranded DNA (ssDNA) by an outer primer. ssDNA serves as a template for DNA synthesis primed by the second inner and outer primers that will then hybridize together at the other end of the target structure. LAMP results in the formation of a stem-loop DNA structure that are, essentially, inverted repeats of the target DNA or RNA sequence. The LAMP reaction may also be accelerated by the addition of two loop primers (one forward and one backward).
LAMP amplified products can be visualized using agarose gel electrophoresis, turbidity, fluorescence, or via colorimetric means. Upon turbidity, the formation of white precipitate caused by magnesium pyrophosphate, a by-product of the reaction, in the amplified mixture is linearly correlated with the amount of DNA synthesized. Turbidity can be quantitatively measured using a turbidimeter, or examined by the naked eye. For electrophoretic, fluorometric, or colorimetric visualization methods, color changes in amplified products can often be detected through simple observation, then quantified.
Variations of LAMP
Visualization of LAMP products. (A) stained with SYBR Green I and observed under natural light (Tube 1: Y. pestis, Tube 2: negative control); (B) with agarose gel electrophoresis (Lane 1: Y. pestis, Lane 2: negative control, Lane 3: DNA ladder marker 100 bp). From: Development and evaluation of loop-mediated isothermal amplification for detection of Yersinia pestis in plague biological samples by Randriantseheno LN, Rahantamalala A, Randrianierenana AL, Rajerison M, Andrianaivoarimanana V. PLoS ONE 15(8). (2020).
- Reverse Transcription LAMP (RT-LAMP) offers a rapid, one-step DNA amplification technique, and has a significantly shortened reaction time due to a bypassed DNA purification step (as required by PCR).
- Proofreading LAMP (PR-LAMP) has been used for genotyping single-nucleotide polymorphisms (SNPs), and utilizes the 3'-5' exonuclease proofreading activity of DNA polymerase. SNPs, in themselves, are variations that occur fairly evenly and frequently across the entirety of the genome. Because of this, they may be commonly targeted as biomarkers for investigating predisposing factors of disease, drug resistance, and pharmacological efficacy. When compared to PCR in combination with restriction fragment length polymorphism (PCR-RFLP), the typing of human genomic samples yielded results that were nearly identical.
- in situ LAMP may be used to provide conclusive identification of some parasites. The high sensitivity of other molecular methods, i.e., PCR, may result in false-positives due to contamination, also known as carry over. Another issue is that the target gene products may be detected even after clearance of the parasites from the subject. In these instances, accurate detection of parasites by microscopic examination is crucial to provide a definitive diagnosis.
- Peptide Nucleic Acid LAMP (PNA-LAMP) uses a genetic mutation as a diagnostic tool and prognostic biomarker to characterize disease. There is potential for PNA-LAMP to be used on whole tumor specimens in an intraoperative, time-sensitive setting where DNA extraction was not possible.
Table 1. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis
| Product ▲ ▼ | Ex (nm)¹ ▲ ▼ | Filter² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 1 mL | 17590 |
| Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 100 µL | 17604 |
| Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 1 mL | 17595 |
| Gelite™ Green Nucleic Acid Gel Staining Kit | 254 nm or 300 nm | Long path green filter | 1 Kit | 17589 |
| Gelite™ Orange Nucleic Acid Gel Staining Kit | 254 nm or 300 nm | Long path green filter | 1 Kit | 17594 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 100 µL | 17700 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 500 µL | 17701 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 1 mL | 17702 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 10 mL | 17703 |
| Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 100 µL | 17704 |
Product Ordering Information
Table 2. Dyes for Loop-Mediated Isothermal Amplification (LAMP)
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 17555 | LAMP Green™ *50X DMSO Solution* | 100 µL |
References
Loop-mediated isothermal amplification of DNA
Loop-Mediated Isothermal Amplification Methods for Diagnosis of Bacterial Meningitis
Loop-Mediated Isothermal Amplification
Homogeneous Assays for Single-Nucleotide Polymorphism Genotyping: Exo-proofreading Assay based on Loop-mediated Isothermal Amplification
Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe
In situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears
Advances in Molecular Diagnosis of Malaria
A Rapid Colorimetric Peptide Nucleic Acid Loop-Mediated Isothermal Amplification Assay for the Detection of the IDH1 Mutation in Glioblastoma