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- ABBREVIATION: Loop-mediated Isothermal AMPlification.
- Diseases are the primary constrains for the development of any living forms i.e., both plants and animals including humans.
- Diagnosis is a critical step for effective treatment and control of infectious diseases.
- PCR (Polymerase Chain Reaction) has revolutionized the earlier detection of infection, tumor, etc.,
- However, LAMP has several pros over PCR.
- LAMP is used in the rapid diagnosis of viral, bacterial and parasitic diseases.
- It helps in the identification of genus and species-specific parasites.
- First reported by Notomi et al in 2000 of EIKEN Chemical Co. Ltd., Japan.
- It is a very sensitive, easy and time-efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.
Image Source: Nature
Amplification of DNA via cyclic and non-cyclic amplification steps using 4 or 6 primers, while the reaction proceeds at a constant temperature using a strand displacement reaction.
Types of Primers used in LAMP
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)
When the target gene (DNA template) and the reagents are incubated at a constant temperature between 60- 65⁰C, the following reaction steps proceed. Stages in Loop-mediated Isothermal Amplification Step 1 One of the LAMP primers can anneal to the complementary sequence of double-stranded target DNA, then initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and releasing a single-stranded DNA.
With the LAMP method, unlike with PCR, there is no need for heat denaturation of the double-stranded DNA into a single strand. The following amplification mechanism explains from when the FIP anneals to such released single-stranded template DNA.
Procedure of LAMP
- It is characterized by the use of the 4 different primers specifically designed to recognize 6 distinct regions on the target gene. In the target gene, the F3c, F2c, and the F1c regions at the 3’ side and the B1, B2, and B3 regions at the 5’ side are the distinct regions
- Amplification and detection of the gene can be completed in a single step, by incubating the mixture of sample primers, DNA polymerase with strand displacement activity and substrate at constant temperature (about 65⁰C).
- It provides high amplification efficiency with DNA being amplified 10⁹-10ⁱ⁰ times in 15- 60 minutes.
- Because of its high specificity, the presence of amplified product can indicate the presence of the target gene.
Amplification: Is of two types
Generation of stem-loop DNA with a dumbbell-shaped structure at both ends.
B. Cycling Amplification:
Dumbbell-shaped DNA is quickly amplified by the use of loop primers.
Loop primers: With the use of Loop Primers, amplification can be achieved within 30 minutes.
- Turbidity – Magnesium Pyrophosphate (white precipitate)
- Fluorescence – Calcein (greenish-yellow)
- Gel Electrophoresis
LAMP vs PCR
|Isothermal Reaction.||Cyclic Reaction.|
|Isothermal Temperature (60-65⁰C).||Variable Temperature.
Denaturation (95⁰C); Annealing (50-60⁰C); Polymerization (72⁰C)
|Doesn’t require expensive
|The detection limit is greater.||The detection limit is lower.|
|Amplification specificity is higher as it uses 4/6 oligonucleotides.||Amplification specificity is lower
than that of LAMP.
|It could be done using crude DNA samples.||Need pure DNA samples for amplification.|
|Loop primers accelerate the reaction rate.||No loop primer.|
|Visualization of DNA could be done through eyes, gel electrophoresis, and turbidimeter.||Visualization of DNA is done through gel electrophoresis.|
To sensitive and prone to contamination and small changes in condition Applications Complicated primer design vDisadvantages
- LAMP is highly sensitive and specific DNA/RNA amplification technique.
- It is a simple, cost-effective technique.
- LAMP is an innovative molecular diagnostic field and can be used for the diagnosis of infectious diseases, food inspection, environmental testing and so on.
- Innovations in biotechnology that combine molecular biology, microfabrication and bioinformatics are moving nucleic acid technologies from futuristic possibilities to common laboratory techniques and modes for diagnoses.
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Loop-mediated Isothermal Amplification (LAMP)