Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction. A strand-displacing DNA polymerase initiates synthesis and 2 specially designed primers form “loop” structures to facilitate subsequent rounds of amplification through extension on the loops and additional annealing of primers. DNA products are very long (>20 kb) and formed from numerous repeats of the short (80–250 bp) target sequence, connected with single-stranded loop regions in long concatamers. These products are not typically appropriate for downstream manipulation, but target amplification is so extensive that numerous modes of detection are possible. Real-time fluorescence detection using intercalators or probes, lateral flow and agarose gel detection are all directly compatible with LAMP reactions. Instrumentation for LAMP typically requires consistent heating to the desired reaction temperature and, where needed, real-time fluorescence for quantitative measurements. Optimized settings for running LAMP experiments on isothermal instruments such as the Axxin T8-ISO and T16-ISO can be found here.
|Reaction Temperature||Amplicon Size||Detection Method(s)|
|65°C||<250 nt||Visual, Lateral flow, Gel, Turbidity|
In addition to the more traditional or complex detection methods, LAMP is so prolific that the products and byproducts of these reactions can also be visualized by eye. For example, magnesium pyrophosphate produced during the reaction can be observed as a white precipitate or added indicators like calcein or hydroxynaphthol blue can be used to signal a positive reaction. Alternatively, using the WarmStart® 2X Colorimetric LAMP Master Mix developed by NEB enables a strong color change from pink to yellow based on a pH change during the reaction. An updated version of this product has been formulated with dUTP and UDG to be compatible with carryover prevention between amplification rounds – WarmStart Colorimetric LAMP 2X Master Mix with UDG. The colorimetric detection technology is a key component of the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit, which can be used in the analysis of SARS-CoV-2, the novel coronavirus that causes COVID-19.
Designing LAMP primers can be challenging, but software tools greatly facilitate this process. We suggest using the NEB LAMP Primer Design Tool to design LAMP primers. After inputting a DNA or RNA sequence of interest, the LAMP Primer Design tool will identify suitable target regions and create the outer F3/B3 and looping inner FIP/BIP primers in a single step. The LoopF/LoopB primers, that accelerate the LAMP reaction, are created in a second step and are strongly recommended for best performance.
LAMP is well-suited for point-of-care and field diagnostics and LAMP assays have been designed for the detection of a wide range of RNA and DNA targets from all manner of sample types. Examples include tests for:
- Rapid detection of coronavirus (COVID-19) RNA
- Direct detection of SARS-CoV-2 RNA from Universal Transport Medium
- Filariasis in humans and insects
- Food and water quality in-field applications
- Detection of the Zika virus in human samples
The LAMP reaction is robust and tolerant of inhibitors, allowing for crude sample prep and minimal nucleic acid purification if desired. WarmStart® RTx and Bst 2.0 WarmStart were developed for optimal performance in LAMP/RT-LAMP and are combined in convenient LAMP Master Mixes to simplify assay design.
- How can I see the products of a LAMP reaction?
- How do I use Tte UvrD Helicase for reducing non-template amplification in LAMP reactions?
- Does NEB have a master mix for LAMP or RT-LAMP reactions?
- What is the difference between Bst DNA Polymerase, Large Fragment, Bst 2.0, and Bst 3.0 DNA Polymerase?
- How do I use WarmStart® RTx in RT-LAMP?
- How do I use Antarctic Thermolabile UDG for carryover prevention in LAMP reactions?
- Loop-mediated Isothermal Amplification (LAMP)
- WarmStart LAMP Kit (DNA & RNA) Protocol (E1700)
- WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800)
- Protocol for LAMP Reactions with Tte UvrD Helicase (#M1202)
- Protocol for unwinding double stranded DNA with Tte UvrD Helicase (#M1202)
- Typical LAMP Protocol (M0275)
- Typical LAMP Protocol (M0538)
- Typical LAMP Protocol (M0374)
- Typical RT-LAMP Protocol
- WarmStart® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) Protocol (NEB #M1708)
- WarmStart® Fluorescent LAMP/RT-LAMP Kit (with UDG) Protocols (NEB #E1708)
- WarmStart Colorimetric LAMP 2X Master Mix with UDG Typical LAMP Protocol (NEB #M1804)
- SARS-CoV-2 Rapid Colorimetric LAMP Detection Assay Protocol (NEB #E2019)
Mitigating Risk and Ensuring Consistent Supply Chain Through Internal SARS-CoV-2 Testing with RT-LAMP
This feature article describes how NEB scientists established a CLIA-certified SARS-CoV-2 onsite testing program utilizing saliva and colorimetric loop-mediated isothermal amplification (LAMP)
- Isothermal Amplification Trifold
- NEB LAMP Primer Design Tool
- Amplification Reagents for Molecular Diagnostics Applications (2017)
- Colorimetric LAMP: Visual Detection for Simple Diagnostics (2017)
- Genome filtering identifies species-specific DNA biomarkers for Mansonella perstans and Mansonella ozzardi, which enable differentiation of these closely related species and other co-endemic filarial parasites (2019)
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NEB® LAMP Primer Design Tool Tutorial
This video explains how to use our LAMP Primer Design web tool, which makes the complex task of LAMP primer design as easy as submitting your sequence.
Loop Mediated Isothermal Amplification (LAMP) Tutorial
Did you know that this isothermal amplification method can be performed in as little as 5-10 minutes with limited resources? Get a quick overview on how LAMP works in this animation.
Behind the Paper: Visual Detection of Isothermal Nucleic Acid Amplification Using pH-sensitive Dyes
One little proton could change the way scientists detect DNA amplification in the field and point-of-care settings. Nathan shares the details of his recent paper.
NEB® TV Ep. 23 – Colorimetric LAMP in point-of-care diagnostics
Learn the basics of Colorimetric LAMP, and how groups like NEB and the World Mosquito Program are using this isothermal amplification method in field tests and point-of-care diagnostics.