Loop-mediated

Isothermal
Amplification
 INTRODUCTION
• LAMP" stands for Loop-mediated Isothermal Amplification.

• This technology was developed by Notomi et al

• It is a very sensitive, easy and time efficient method

• LAMP is an isothermal nucleic acid amplification technique. In contrast to the

polymerase chain reaction (PCR) technology, isothermal amplification is carried out at
a constant temperature, and does not require a thermal cycler.

• It is characterized by the use of the 4 different primers specifically designed to

recognize 6 distinct regions on the target gene.
Principles of LAMP

• The LAMP method relies on the activity of a strand-displacing DNA

polymerase and a set of four to six specially designed primers that
recognize six to eight distinct regions of the target sequence. The
amplification process is isothermal, typically performed at 60–65°C,
eliminating the need for expensive thermal cyclers.
Lamp Detection

• In a LAMP assay, the reaction takes place in a single tube containing buffer,
target DNA, DNA polymerase and primers.

• The tube is incubated at 64°C in a regular laboratory water bath or heat

block that helps in maintaining a constant temperature.

• The amplified product can be detected by naked eye as a white precipitate

or a yellow-green color solution after addition of SYBR green to the reaction
tube
 Key Features of LAMP
• High Specificity: Multiple primers targeting distinct regions of the DNA
ensure specificity.

• Rapid Amplification: Amplification is completed within 30–60 minutes.

• Robustness: Works well with crude samples, reducing the need for extensive
sample preparation.

• Visual Detection: Amplification can be detected visually using dyes or

turbidity.
Types of Primers
• LAMP is characterized by the use of 4 different primers
specifically designed to recognize 6 distinct regions of the target
gene.

• The four primers used are as follows

 Forward Inner Primer (FIP):
 Forward Outer Primer (FOP): The FOP (also called F3 Primer)
 Backward Inner Primer (BIP):
 Backward Outer Primer (BOP):
 Loop primers
• The loop primers(either loop primer B or loop primer F) containing sequences
complementary to the single stranded loop region( either between the B₁ and B₂ regions or
between F₁ and F₂ regions) on the 5’ end of the dumbell like structure, provide an increased
number of the starting points for the LAMP method.

• The investigation on how Loop Primers affect amplification time (original

method: no Loop Primer; rapid method: with Loop Primers) shows that the
time required for amplification with Loop Primers is one-third to one-half of
that without Loop Primer. With the use of Loop Primers, amplification can
be achieved within 30 minutes.
Advantages

• Isothermal Conditions: Simplifies instrumentation, reducing costs.

• Rapid Results: Faster than PCR, with results in under an hour.

• High Sensitivity: Detects as few as 6–10 copies of target DNA.

• Robustness: Effective with crude samples, such as blood, saliva, or urine.

• Ease of Use: Simple protocols enable use in resource-limited settings.

 Limitations
• Primer Design: Requires careful design of multiple primers, increasing complexity.

• Complicated primer design which is to sensitive and prone to contamination and

small changes in condition suffers from non-specific binding through formation of
form primer dimers.

• The use of multiple primers increases the risk of primer–primer hybridizations

resulting in template‐free amplification, leading to False Positives

• Quantification: Less suited for quantitative analysis compared to qPCR.

Steps of LAMP
Initiation:
• The forward and backward inner primers (FIP and BIP) bind to their
complementary sequences, initiating strand displacement by the polymerase.

Cycling Amplification:
• The displaced single-stranded DNA forms stem-loop structures due to self-
annealing of the primers.
• Outer primers (F3 and B3) displace the inner primers, generating a cascade of
strand-displacement reactions.
Steps of LAMP

Exponential Amplification:
• The process amplifies the target DNA in a continuous loop, producing
cauliflower-like DNA structures with multiple loops.

Detection:
• Amplified products are detected by various methods, such as turbidity,
fluorescence, or color changes
 Types of LAMP Detection
Colorimetric LAMP
• Principle: Changes in pH during amplification alter the color of a pH-sensitive dye
(e.g., phenol red or hydroxy naphthol blue).
• Application: Rapid visual detection without requiring specialized equipment.

Turbidimetric LAMP
• Principle: Magnesium pyrophosphate, a by-product of DNA synthesis, precipitates
during amplification, increasing turbidity.
• Application: Quantitative measurements using a turbidimeter or visual assessment. .
 Types of LAMP Detection
Fluorescent LAMP
• Principle: Fluorescent dyes (e.g., SYBR Green or EvaGreen) intercalate into double-
stranded DNA, emitting light upon binding.
• Application: Real-time monitoring and quantification of DNA

Lateral Flow LAMP

• Principle: Biotin- or fluorescein-labeled amplicons are detected on a
lateral flow strip using complementary probes.
• Application: Portable diagnostic tests for field settings
 Types of LAMP Detection
CRISPR-Cas LAMP
• Principle: LAMP products activate CRISPR-associated nucleases (e.g., Cas12 or
Cas13), which cleave reporter molecules for signal generation.
• Application: Ultra-sensitive and specific detection for pathogens and genetic
mutations.

Paper-Based or Microfluidic Systems

• Principle: Integrates LAMP reactions with paper or microfluidic devices for miniaturized detection.

• Technique: Color changes, fluorescence, or electrochemical signals.

• Advantages: Portable and scalable for low-resource settings.

• Applications: Point-of-care diagnostics, environmental testing.

Types of LAMP Detection

Nanomaterial-Based Detection
• Principle: Uses nanoparticles (e.g., gold nanoparticles) that interact with
amplified DNA products.
• Technique: Color changes (e.g., aggregation of gold nanoparticles) or plasmonic
shifts.
• Advantages: Sensitive and versatile.

• Applications: Pathogen detection, genetic studies.

Improvement/Advancement in LAMP
Improvement of LAMP
• DNA polymerase
o Bst DNA polymerase and its engineered variants like Bst 2.0 and Bst 3.0
o OmniAmp polymerase (OmniAmp pol)
• Primer of LAMP
 Primers for higher speed and higher sensitivity
 Primers for lower reaction temperature
 Primers for amplifying short gene sequences
• Combination of LAMP with other techniques
 Loop-mediated isothermal amplification with RCA
 Loop-mediated isothermal amplification with RPA
 Loop-mediated isothermal amplification with HCR
 Loop-mediated isothermal amplification with CRISPR-Cas
 Loop-mediated isothermal amplification with biosensors
 Loop-mediated isothermal amplification with microfluidic technology
1. Advances in Enzyme
Development
• Bst DNA Polymerase Enhancements:
• Bst DNA polymerase remains the cornerstone of LAMP, but
variants like Bst 2.0 and Bst 3.0 have significantly improved
its capabilities.
• Bst 2.0 offers increased polymerization speed, thermal
stability, and reduced susceptibility to inhibitors, making it
suitable for impure samples.
• Bst 3.0 integrates reverse transcriptase activity, allowing for
RNA amplification, and demonstrates remarkable efficiency
even in challenging conditions
• OmniAmp Polymerase:
• Sourced from thermophilic viral metagenomes, OmniAmp
polymerase outperforms traditional enzymes in speed and
strand displacement efficiency.
• It enables amplification within 30 minutes and maintains
robust performance in high-inhibitor environments​
• FEN1-Bst DNA Polymerase:
• A novel recombinant enzyme combining DNA synthesis,
strand displacement, and cleavage functions, FEN1-Bst
enhances detection limits, making it suitable for applications
requiring ultra-sensitivity
2. Improved Primer Design

• Incorporation of Loop Primers:

• Introduced to accelerate amplification by targeting stem-loop regions, loop primers reduce
reaction times by 30–50%.
• These primers are particularly effective for detecting pathogens like Clostridium
difficile and Listeria monocytogenes​
• Stem Primers:
• Developed as an alternative to loop primers, stem primers offer greater design flexibility and
similar performance.
• They can work in tandem with loop primers, enhancing the efficiency and sensitivity of the
LAMP reaction​
• Degenerate Primers:
• Allow consensus detection of multiple pathogen species by accommodating sequence
variability.
• Useful in screening assays for diverse infections, such as pulmonary mycobacterial species​
• Phosphorothioated Primers:
• Enable LAMP reactions at lower temperatures (~40°C), maintaining sensitivity while
expanding usability
3. Enhanced Detection Methods
• Fluorescent Indicators:
• Intercalating dyes like SYBR Green and SYTO enable real-time
monitoring with high sensitivity.
• These methods are useful for clinical applications requiring real-
time quantification​
• Colorimetric Indicators:
• pH-sensitive dyes and metal-binding indicators (e.g., hydroxy
naphthol blue and calcein) provide visible color changes.
• These techniques simplify LAMP for field and point-of-care testing​
• Electrochemical Sensors:
• Emerging integration with biosensors enhances detection precision
and enables multiplexing capabilities
4. Automation and
Miniaturization
• Microfluidic Integration:
• LAMP has been adapted into microfluidic systems for
automation, reducing reagent consumption and enabling
high-throughput analysis.
• Portable devices with integrated fluid handling simplify
diagnostics in remote areas​
• Point-of-Care Testing:
• Smartphone-based platforms allow real-time LAMP
monitoring, bringing advanced diagnostics to resource-limited
settings
5. Expanded Applications
• Dual-Temperature and RNA Detection:
• The development of enzymes with reverse transcriptase
activity, such as Bst 3.0, has enabled LAMP-based RNA virus
detection (e.g., SARS-CoV-2, Foot-and-Mouth Disease Virus).
• These improvements enhance the utility of LAMP for emerging
infectious diseases​
• Multiplex Detection:
• Advanced primer designs and signal transduction methods
allow for the simultaneous detection of multiple targets in a
single reaction.
• Applications include pathogen detection in food safety and
environmental monitoring​
Detection of human diseases
• LAMP technology has been increasingly utilized for the rapid detection
of human diseases, including:
1.Hand, Foot, and Mouth Disease (HFMD): Zhang et al. (2024)
developed a multiplex LAMP assay that detects multiple enteroviruses
(Human enterovirus 71, Coxsackievirus A16, A6, and A10) in clinical
samples within 30 minutes, with 100% sensitivity and specificity.
2.Severe Fever with Thrombocytopenia Syndrome (SFTS): Tian et
al. (2023) evaluated LAMP for on-site diagnosis of SFTS, showing a
sensitivity of 81.9% and a specificity of 96.3% using 279 plasma
samples from patients.
3.Tuberculosis (TB): Yang et al. (2023) developed a sensor combining
LAMP with lateral flow immunoassay technology, capable of detecting
TB within 80 minutes, with a limit of detection (LOD) of 100 fg/reaction.
a. Malaria Detection
• Study: A study compared LAMP to microscopy and PCR for
detecting Plasmodium falciparum.
• Findings: LAMP showed higher sensitivity (98%) compared to
microscopy (86%) and was more field-friendly than PCR.
• Applications: Used in malaria-endemic regions for rapid
diagnosis.
b. Tuberculosis Detection
• Study: Eiken Chemical developed a LAMP kit for Mycobacterium
tuberculosis detection.
• Findings: LAMP outperformed smear microscopy in sensitivity
and required minimal technical expertise.
• Applications: Implemented in low-resource settings. .
C. COVID-19 Testing
• Study: LAMP assays targeting SARS-CoV-2 genes (e.g., ORF1ab, N,
and E genes) were validated.
• Findings: Provided comparable sensitivity to RT-PCR within 30
minutes.
• Applications: Widely used for rapid screening during the
pandemic.
d. Dengue and Zika Virus Detection
• Study: Reverse transcription LAMP (RT-LAMP) was used to detect
RNA of dengue and Zika viruses.
• Findings: High sensitivity and specificity, with colorimetric
detection simplifying field use.
• Applications: Early diagnosis during outbreaks in tropical regions.