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.
Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification used as an alternative to polymerase chain reaction (PCR)
. LAMP has been used as a rapid, accurate, and efficient method for the detection and identification of infections including those that are bacterial, viral, fungal, and parasitic in nature for humans, animals, and plants. Notably, some RNA viruses like HIV-1, Dengue virus, SARS-CoV-2
, and Ebola (EBOV), as well as the bacterial disease, meningitis, have been detected through LAMP techniques.
LAMP has widely been used as a rapid, diagnostic tool to ensure timely clinical treatment and prevention of disease outbreaks. The LAMP protocol is incredibly simple; all it requires are at least 4 suitable primers
(often 6), a DNA polymerase, and a regular laboratory water bath, heat block, or oven for processing. LAMP can be used as a powerful point of care device and with variations in the technique the availability for high throughput screening has increased.
LAMP vs. PCR
LAMP techniques work
|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|
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
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
Variations of LAMP techniques have been created that couple other research methods for more precise and accurate analysis. To date, only a handful of LAMP assays have been approved for clinical use due to, mainly, a low tolerance to highly variable target sequences, non-specific amplification, and the limitations associated with multiplexing. These variations in the LAMP technique are limited in uniformity as they are most commonly practiced in research
, though numerous studies exist that provide evidence of how LAMP has the potential to advance clinical diagnostics.
- 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
|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)