Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that enables the rapid and specific replication of target DNA sequences under isothermal conditions, typically at 60–65°C, without the need for thermal cycling equipment. Developed as an alternative to polymerase chain reaction (PCR), LAMP utilizes a set of four to six primers that recognize six to eight distinct regions on the target DNA, along with a strand-displacing DNA polymerase such as Bst polymerase to generate up to 10⁹ copies of the target in under an hour.[1] This method produces characteristic stem-loop DNA structures and cauliflower-like multimers, allowing for simple detection through turbidity, fluorescence, or colorimetric indicators.[1] Invented in 2000 by Notomi et al. at Eiken Chemical Company in Japan, LAMP was designed to overcome limitations of earlier isothermal methods like nucleic acid sequence-based amplification (NASBA) and self-sustained sequence replication (3SR), which suffered from lower specificity or efficiency at constant temperatures.[1] The technique's core mechanism begins with an inner primer hybridizing to the target DNA, initiating strand displacement synthesis by the polymerase, which releases a displaced strand that forms a loop structure; subsequent cycling of this process, often accelerated by loop primers, leads to exponential amplification.[2] Its high specificity stems from the multiple primer recognitions, reducing the risk of non-specific amplification compared to PCR, while the isothermal nature makes it ideal for resource-limited settings.[2] LAMP has become a cornerstone in molecular diagnostics, particularly for detecting pathogens in infectious diseases such as malaria, tuberculosis, and COVID-19, enabling point-of-care testing in field environments like clinics or even the International Space Station.[2] Advantages include its cost-effectiveness (requiring only a heat block or water bath), high sensitivity (detecting as few as 10 copies of template DNA), and adaptability for RNA targets via reverse transcription (RT-LAMP).[2] Recent advancements, spurred by the COVID-19 pandemic, have integrated LAMP with CRISPR-Cas systems for enhanced detection and multiplexed assays, further expanding its utility in global health surveillance and food safety monitoring.[3] Despite challenges in primer design and potential aerosol contamination, LAMP's robustness and simplicity continue to drive its adoption in over 10,000 peer-reviewed studies since its inception.[2]History and Development
Invention and Early Work
Loop-mediated isothermal amplification (LAMP) was invented by Toshitsugu Notomi and colleagues at Eiken Chemical Company, Ltd., in Tokyo, Japan.[4] The technique emerged from efforts to create a nucleic acid amplification method that operates under isothermal conditions, eliminating the need for complex thermal cycling equipment typically required in polymerase chain reaction (PCR).[4] The method was first publicly described in a seminal 2000 paper published in Nucleic Acids Research, titled "Loop-mediated isothermal amplification of DNA."[4] In this work, Notomi et al. outlined LAMP as a process that uses a set of four specially designed primers and a DNA polymerase with strand displacement activity to produce concatenated DNA structures with repeatedly integrated sequence regions.[4] The primary motivation was to develop a simple, rapid amplification technique suitable for diagnostics in field settings or resource-limited environments, where access to thermal cyclers is impractical.[4] This addressed the limitations of existing methods by enabling high-specificity amplification at a constant temperature, typically around 60–65°C.[4] Early experiments in the 2000 study demonstrated LAMP's specificity through the use of multiple primers that recognize six distinct sequences on the target DNA, minimizing non-specific amplification.[4] Sensitivity was shown by detecting as few as six copies of target DNA in a 45-minute reaction, outperforming conventional PCR in speed and yield under isothermal conditions.[4] These tests employed Bst DNA polymerase, derived from Bacillus stearothermophilus, prized for its robust strand displacement activity that facilitates continuous amplification without denaturation steps.[4] Preceding the publication, Eiken Chemical Company filed a patent application for the LAMP method in Japan in November 1998 (JP2000283862), securing intellectual property that facilitated its subsequent commercialization as a diagnostic tool.[5] This filing marked the foundational step toward making LAMP accessible for practical applications beyond research.[5]Key Milestones and Evolution
In 2002, shortly after the initial description of loop-mediated isothermal amplification (LAMP), researchers introduced loop primers to enhance the method's efficiency. Developed by Nagamine et al., these additional primers bind to newly formed stem-loop structures during amplification, facilitating faster strand displacement and reducing reaction times from over 60 minutes to approximately 30 minutes or less.[6] In 2004, the technique was advanced to reverse transcription LAMP (RT-LAMP) for direct detection of RNA targets such as viral genomes, with a 2006 application enabling rapid identification of pathogens like avian influenza virus H5, with reactions completing in under 60 minutes at constant temperature.[7][8] Commercialization began in the early 2000s, with Eiken Chemical Co., Ltd. launching the first Loopamp kits in 2002 for bovine embryo sexing and in 2003 for SARS-CoV detection. Subsequent releases expanded to diagnostic tools for tuberculosis in 2011 and malaria in 2012, while companies like New England Biolabs introduced WarmStart LAMP kits in the late 2010s, providing user-friendly reagents for both DNA and RNA amplification.[9][10][11] During the 2010s, LAMP evolved toward multiplexing to detect multiple targets simultaneously, addressing needs in complex diagnostics like bacterial identification. For instance, assays detecting Salmonella serovars and other pathogens in a single reaction emerged around 2017, improving throughput without compromising specificity. Integration with biosensors further advanced the method, enabling automated, portable detection through electrochemical or optical readouts for on-site applications.[12][13] The COVID-19 pandemic in 2020 propelled LAMP's global adoption for SARS-CoV-2 detection, with RT-LAMP assays developed as early as February that year offering results in 30-60 minutes using minimal equipment. These point-of-care tests gained traction in resource-limited settings, aligning with World Health Organization guidelines for accessible molecular diagnostics during outbreaks.[14] Parallel to these developments, LAMP transitioned from laboratory-based protocols to portable formats by the mid-2010s, incorporating battery-powered heaters and simple incubators for field use. Devices like compact, solar- or battery-operated systems emerged around 2015, supporting isothermal reactions in remote environments for pathogen surveillance.Principle and Mechanism
Primer Design and Recognition
Loop-mediated isothermal amplification (LAMP) relies on a specialized set of primers that recognize multiple distinct regions of the target DNA, typically 6 to 8 sites, to achieve high specificity and enable continuous amplification under isothermal conditions. Unlike polymerase chain reaction (PCR), which uses only two primers, LAMP employs four to six primers, including two outer and two inner primers as the core set, with optional loop primers for enhanced efficiency. This multi-primer approach ensures that amplification occurs only when all recognition sites match the target sequence, minimizing non-specific products.[15] The core primers consist of the forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), and backward outer primer (B3). The FIP is a composite primer approximately 40-49 nucleotides long, comprising a forward 1 complementary sequence (F1c, about 20-25 nt) connected via a non-complementary TTTT linker to a forward 2 sequence (F2, about 15-25 nt), which anneals to the F2c region on the target; similarly, the BIP (40-49 nt) includes the backward 1 complementary (B1c) and backward 2 (B2) sequences targeting B1c and B2c regions. The outer primers, F3 and B3, are shorter at 16-21 nt and bind to the F3c and B3c regions, respectively, facilitating initial strand displacement. These primers target six distinct regions (F3, F2, F1, B1, B2, B3) on the template DNA, with the inner primers' dual structure allowing formation of loop structures during amplification.[15] Optional loop primers, LF (loop forward) and LB (loop backward), each 18-25 nt long, can be added to further accelerate the reaction by annealing to single-stranded loops formed between the F1/F2 and B1/B2 regions, respectively, thereby increasing the priming sites and reducing reaction time by up to 50%. Introduced to enhance cycling efficiency, these primers target two additional regions, bringing the total to eight recognition sites when both are used.[16] Primer design emphasizes avoiding secondary structures, primer-dimer formation, and non-specific binding, with typical melting temperatures (Tm) of 60-65°C for compatibility with the isothermal reaction. Specialized software such as PrimerExplorer, developed by Eiken Chemical Company, automates the selection of primer sets by analyzing target sequences for optimal spacing (e.g., 120-150 bp between F1 and B1 regions) and compatibility, ensuring high specificity.[17] The multi-site recognition by LAMP primers confers greater specificity than PCR's two-primer system, as mismatched amplification requires errors at multiple independent sites, effectively suppressing off-target products even in complex samples.[15]Amplification Process and Strand Displacement
Loop-mediated isothermal amplification (LAMP) operates under isothermal conditions, typically at 60–65°C, eliminating the need for thermal cycling equipment required in methods like PCR. This process relies on the strand displacement activity of Bst DNA polymerase, a thermostable enzyme derived from Bacillus stearothermophilus, which synthesizes new DNA strands while displacing downstream strands without requiring a separate helicase for unwinding double-stranded DNA. The reaction initiates and sustains amplification through primer-mediated strand invasion and displacement, enabling continuous DNA synthesis in a single step. Unlike PCR, which depends on repeated denaturation, annealing, and extension phases, LAMP achieves self-sustained amplification via these displacement events, producing results in 30–60 minutes. The amplification begins with the inner primers, FIP and BIP, which anneal to their complementary sequences (F2c and B2c, respectively) on the target DNA. Bst polymerase extends from these primers, initiating complementary strand synthesis. The outer primers, F3 and B3, then anneal to the F3c and B3c regions and initiate strand displacement synthesis, displacing the adjacent strands and generating long single-stranded templates. This displacement creates the foundation for subsequent primer binding. The process is highly specific due to the recognition of six distinct sequences on the target DNA by the primer set. In the subsequent phase, extension from FIP on one strand produces a structure with a 5'-end loop, while BIP extension on the complementary strand, aided by further displacement, forms a dumbbell-shaped DNA molecule with inverted repeats at both ends. These stem-loop structures serve as templates for further amplification, where continued strand displacement by Bst polymerase elongates the stems, maintaining the loop configuration. The addition of loop primers (forward and backward) accelerates the reaction by annealing to the single-stranded loop regions of the dumbbell structures, facilitating rapid primer invasion and multiple initiation points for new strand synthesis. This enables continuous displacement and exponential amplification, as each cycle generates additional templates for primer binding. The loop primers, while optional, significantly enhance the reaction kinetics by promoting strand invasion at non-terminal sites. The overall process results in a mixture of concatenated, dumbbell-shaped DNA products featuring multiple loops, often described as cauliflower-like structures, yielding approximately 10^9 copies of the target sequence in less than 60 minutes under standard conditions. This exponential growth in LAMP can be conceptually modeled as following a pattern similar to iterative doubling, where the number of amplicons increases rapidly due to the multiple primer sets and continuous cycling, though the exact kinetics depend on primer concentrations and reaction conditions. The absence of a denaturation step ensures the reaction remains isothermal and robust, making LAMP suitable for point-of-care applications.Reaction Setup and Procedure
Components and Reagents
The loop-mediated isothermal amplification (LAMP) reaction requires a precisely formulated mixture of components to enable efficient, strand-displacing DNA synthesis under isothermal conditions. The core elements include the DNA template, primers, deoxynucleotide triphosphates (dNTPs), a specialized DNA polymerase, buffer salts, and betaine, with additional enzymes for reverse transcription in RNA-targeted variants (RT-LAMP). Typical reaction volumes are 25 µL, and concentrations are optimized for high yield and specificity. The DNA template serves as the starting material for amplification, typically 0.1–10 ng per reaction (equivalent to ~10²–10⁵ copies for a 1 kb target), with detection sensitivity down to ~10 fg, and can be single-stranded or denatured double-stranded to facilitate initial primer binding.[18] Primers are crucial for specificity, consisting of four to six oligonucleotides: inner primers (forward inner primer, FIP; backward inner primer, BIP) at 0.8-1.6 µM each to initiate strand displacement, outer primers (F3 and B3) at 0.2 µM each for initial template recognition, and optional loop primers (LF and LB) at 0.2 µM to 1.6 µM to accelerate cycling by annealing to newly formed loops. dNTPs provide the nucleotide building blocks for DNA elongation, supplied at a total concentration of 0.8-1.4 mM (equimolar for each dATP, dCTP, dGTP, and dTTP). The polymerase, typically Bst DNA polymerase (large fragment) or its variants like Bst 2.0, is included at 8-16 units per 25 µL reaction due to its high strand displacement activity, which allows continuous amplification without thermal cycling. The reaction buffer maintains optimal ionic conditions, commonly comprising 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2-8 mM MgSO₄ as a cofactor for polymerase activity, and 0.1% non-ionic detergent (e.g., Triton X-100 or Tween 20) to stabilize the enzyme. Betaine is added at 0.8-1.2 M to minimize secondary structure formation in GC-rich templates, enhancing amplification efficiency by equalizing the melting temperatures of AT- and GC-paired regions. For RT-LAMP assays targeting RNA, a reverse transcriptase such as avian myeloblastosis virus (AMV) RT is incorporated at 0.5-5 units per reaction to generate complementary DNA from the RNA template prior to LAMP amplification. Optional additives, such as fluorescent dyes (e.g., SYBR Green) or colorimetric indicators (e.g., hydroxy naphthol blue), may be included at low concentrations (typically 0.1-1 µM) to enable post-amplification detection without interfering with the core reaction.[18]Step-by-Step Protocol
The standard loop-mediated isothermal amplification (LAMP) protocol involves several key steps to ensure reliable amplification of target nucleic acids under isothermal conditions. This procedure is designed for simplicity, requiring minimal equipment such as a heat block or water bath, and typically yields results within an hour.[19] Step 1: Primer Design, Synthesis, and ValidationLAMP requires a set of four to six primers that recognize six to eight distinct regions on the target sequence to achieve high specificity and form loop structures during amplification. Primers include outer primers (F3 and B3) and inner primers (FIP and BIP), with optional loop primers (LF and LB) to accelerate the reaction. Use specialized software such as PrimerExplorer (Eiken Chemical Co.) to design primers based on the target sequence, ensuring optimal melting temperatures (typically 60–65°C for F3/B3 and 5–6°C higher for FIP/BIP) and avoiding self- or cross-dimerization. Synthesize primers commercially and validate them experimentally by testing amplification efficiency with known positive templates via gel electrophoresis or real-time turbidity monitoring; adjust designs if non-specific products appear.[20][19] Step 2: Reagent Preparation and Reaction Mixture Assembly
Prepare a master mix in a sterile microtube to minimize pipetting errors and contamination risk. For a standard 25 μL reaction volume, combine 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3 (and 0.8–1.6 μM each of loop primers if used), 1.4 mM dNTPs, 0.8 M betaine (to reduce secondary structure formation), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 6–8 mM MgSO₄ (as Mg²⁺ cofactor for polymerase), 0.1% Tween 20 (for enzyme stability), 8 units of Bst DNA polymerase (large fragment with strand displacement activity), and 1–5 μL of template DNA (10–100 fg to 10 ng, depending on purity). Add distilled water to reach the final volume; for RNA targets, include 0.625 units of AMV reverse transcriptase in the mix for reverse transcription. Vortex gently and briefly centrifuge to collect the mixture at the bottom of the tube.[20][19] Step 3: Incubation
Place the reaction tube in a heat block, water bath, or isothermal device preheated to 60–65°C, as this temperature range optimizes Bst polymerase activity while enabling primer annealing and strand displacement. Incubate for 30–60 minutes; shorter times (e.g., 30 minutes) suffice for high-template loads, while longer incubations improve sensitivity for low-abundance targets. No thermal cycling equipment is required, making LAMP suitable for field applications. Monitor progress optionally via real-time turbidity if using a compatible instrument, where an increase in optical density at 400 nm indicates amplification.[20][19] Step 4: Optional Termination
Following incubation, heat the reaction at 80°C for 2 minutes to inactivate the polymerase and halt amplification, preventing non-specific extension during storage or analysis. This step is recommended for consistency, especially in multi-sample workflows.[20][19] Troubleshooting
If amplification yield is low, optimize Mg²⁺ concentration (test 4–10 mM increments) as it influences polymerase activity and primer binding; excessive Mg²⁺ can promote non-specific products. For crude samples like blood or soil extracts, inhibitors (e.g., heme or humic acids) may reduce efficiency—dilute the template 10–100-fold or perform a simple purification step to mitigate this. Always include no-template controls to detect contamination.[21][19] Variations
For one-pot reverse transcription LAMP (RT-LAMP), incorporate reverse transcriptase into the initial master mix and proceed directly to incubation at 60–65°C for 30–60 minutes, enabling simultaneous reverse transcription and amplification of RNA targets without separate steps. This variation is widely used for viral diagnostics.[20] Safety Considerations
Wear gloves throughout to prevent nuclease contamination and personal exposure to reagents; dispose of amplicon-containing waste as biohazardous material. To avoid carryover contamination from previous reactions, decontaminate workspaces and pipettes with 10% bleach or UV irradiation (e.g., 254 nm for 15–30 minutes on exposed surfaces), as LAMP amplicons are stable and highly amplifiable.[19][22]