Loop-Mediated Isothermal Amplification (LAMP): Principle, Mechanism, and Applications

Loop-mediated isothermal amplification (LAMP) is one of the advanced molecular biology techniques that offers accurate, rapid, and cost-effective diagnosis of infectious diseases. Primarily, this technique is conducted where the PCR facilities and infrastructures are unavailable. The amplification of nucleic acid in the LAMP technique is based on strand displacement reaction and a stem-loop structure formation under isothermal conditions.

This makes LAMP well suited to testing at the point of care, in clinics and field settings that have no thermocycler and little laboratory infrastructure. During the COVID-19 pandemic, for example, RT-LAMP assays were used to detect SARS-CoV-2 directly from saliva or swab samples with a simple heat source and a visible color change.

Background

Large varieties of diagnostic methods have been developed and implemented since the discovery of diseases. However, many pathogens still survive in the world without being eliminated. Diagnosis, therefore, plays a critical role in the effective treatment and control of infectious diseases, and the diagnostic methods vary with time, from traditional to advanced ones. The conventional ways of diagnosis include culture method, microscopy, and biochemical tests. The culture method is still a core technology in the clinical laboratory. It gives information about the viability of the pathogens and further tests, like antibiotic sensitivity tests and biochemical tests, which are done on their basis. But, while culturing pathogens, the slow multiplication of microbes causes a delay in diagnosis, and difficulty in selective cultivation may create problems in diagnosis.

On the other hand, direct microscopy is frequent, rapid, and simple, but the poor sensitivity of smear tests may result in false negativity. Furthermore, mixed Infections with two or more species are usually not recognized in such methods. The newer and advanced technologies, like serology and antigen capture tests, are rapid but may lack sensitivity and specificity.

Delay in diagnosis delays the therapy, exacerbating the severity of diseases. Similarly, a wrong diagnosis may lead to inappropriate medicine prescription, affecting treatment and creating resistant microorganisms.

In traditional methods, confusion is often caused by variations in chemical composition and morphology. The advanced technologies based on the DNA of pathogenic cells are highly effective in overcoming these challenges. DNA is more informative than the chemical composition of pathogens and can be extracted. PCR is a molecular technique that requires expensive equipment and is more time-consuming.

Although PCR was a revolution in diagnosis, it is costly and time-consuming. So, an alternative isothermal amplification (LAMP) was introduced. Compared with PCR, LAMP requires less time and is less expensive. Hence LAMP has excellent potential in diagnosis.

LAMP technology was first reported in 2000 by Notomi et al. of Japan. As the name suggests, LAMP amplifies a target DNA segment under isothermal conditions, and loop structures are formed when the LAMP primers amplify their target DNA sequence. The highly efficient polymerase enzyme used amplifies the target DNA sequence in a minimal amount of sample, producing millions of copies of the target sequence.

Principle of LAMP

LAMP answers one practical question: how do you copy a specific piece of DNA millions of times without a machine that heats and cools the sample over and over?

Ordinary PCR needs a thermocycler because it separates the two DNA strands by heating to about 95 °C, then cools to let primers bind, then warms again to extend them, repeating the cycle 30 or more times. The heating step is what pulls the double helix apart.

LAMP removes the need for that heating in two ways.

1. First, it uses a different enzyme. The polymerase in LAMP is Bst DNA polymerase, from Bacillus stearothermophilus. Its special property is strong strand displacement: as it synthesizes a new strand, it pushes the old complementary strand out of the way. Because the enzyme itself separates the strands as it goes, the reaction never needs a high-temperature denaturation step. Everything happens at one constant temperature, about 60 to 65 °C.
2. Second, it uses a clever primer design that makes the DNA fold back on itself into loops. This is the part worth understanding, because it is the whole idea behind the name.

LAMP uses four core primers that recognize six separate regions of the target. Two of them, the inner primers FIP and BIP, are unusual: each one is really two sequences joined together. FIP carries an F2 segment at its 3′ end and an F1c segment at its 5′ end. When FIP primes synthesis, the new strand it makes ends up carrying both an F1 region and, further along, a sequence complementary to it (F1c). Because those two regions are complementary and sit on the same strand, that strand folds back and pairs with itself, forming a loop. The same thing happens at the other end with BIP.

The result is a DNA molecule with a loop at each end, shaped like a dumbbell. That dumbbell is the key intermediate. Its loops give the primers ready-made single-stranded landing sites, so new rounds of synthesis can start again and again without any heating to reopen the DNA. The reaction becomes self-sustaining, and copies pile up extremely fast, up to a billion in under an hour.

So the principle in one line: a strand-displacing enzyme removes the need to heat, and a self-looping primer design keeps the reaction cycling on its own at one temperature.

Mechanism of LAMP

Target DNA from pathogen cells is amplified by employing Bst DNA polymerase (isolated from Bacillus stearothermophilus). A set of 4-6 specially designed primers hybridize into six or eight different parts of the target DNA sequence. The DNA polymerase undergoes strand displacement activity along with two inner primers and two outer primers in both the forward and backward direction of the target DNA, thereby initiating the synthesis. Two additional primers, which are specially designed to anneal at the loop structure, can facilitate subsequent rounds of amplification and enhance the sensitivity of the LAMP reaction.

The types of primers required are; forward inner primer (FIP), forward outer primer (FOP or F3), backward inner primer (BIP), and backward outer primer (BOP or B3), forward loop primer (LF), and backward loop primer (LB).

Figure: Loop-mediated isothermal amplification (Source: Trends in Parasitology)

DNA amplification in LAMP can be divided into three steps illustrated in the figure. The three steps are:

Starting structure producing phase

• Firstly, the DNA sequence of interest is identified with three forward target regions and three backward target regions; primers are designed according to the target sequence based on the conserved region.
• Then, based on each sequence of six target regions, LAMP primers are designed.
• Now, denaturation of the DNA containing the target is done to separate two complementary strands.
• After that, FIP (forward inner primer) binds to the F2c region through its F2 segment and primes synthesis, and the polymerase extends the new strand in the 5′ to 3′ direction.
• Similarly, the forward outer primer, F3, now binds to a region upstream from the FIP binding site.
• Thus the newly formed strand, created by the action in FIP, is now unzipped by the F3 primer as it synthesizes complementary DNA.
• Similarly, the backward inner primer (BIP) binds the newly made strand at the B2c region, and synthesis again proceeds in the 5′ to 3′ direction.
• Now, the backward outer primer, B3, binds upstream from the binding site of BIP.
• B3 primer now unzips the new strand, which was formed by the action of BIP as it synthesizes complementary DNA.
• Finally, the newly synthesized strand carries two pairs of complementary regions on the same strand, F1 with F1c and B1 with B1c. Because these regions are complementary and lie on one strand, the strand folds back and pairs with itself at each end, forming a dumbbell-shaped structure with a loop at each end.

Cycling amplification step

• Here, the looped structure forms the basis for the LAMP auto cycling process.
• Then FIP and BIP bind at F2c and B2c respectively, self-priming through the F1-F1c and B1-B1c complementary regions occurs, and the reaction continues cycling through further rounds of synthesis.
• In addition, forward and backward loop primers (LF and LB) provide other priming sites and increase the amplification rate.
• The binding site for LF is a sequence between F1 and F2 regions of the target, whereas that for LB is a sequence between B1 and B2 target regions.

Elongation step

• All six primers’ activity produces much different-sized DNA with multiple repeats of the initial target sequence.
• Long DNA products, more than 20 kbp, are formed from numerous repeats of the short (80-250 bps) target sequence. These are connected with single-stranded loop regions in long concatemers.
• The addition of reverse transcriptase amplifies DNA from RNA sequences (RT-LAMP), making diagnosis possible from RNA samples.
• Although the downstream manipulation is not suitable, the target amplification is extensive enough to perform numerous modes of detection.

Concatemers: One of two or more DNA or RNA molecules that are covalently joined (end to end) in the same orientation.

LAMP versus PCR

LAMP and PCR both copy a target DNA sequence millions of times, but they work differently and suit different settings. The table below sets out the main differences.

The practical takeaway: PCR remains the reference method in a well-equipped laboratory, especially where accurate quantification or multiplexing is needed. LAMP wins where speed, simplicity, and no thermocycler matter most, which is why it has become a leading choice for point-of-care and field diagnosis. The higher primer count is the reason for LAMP's very high specificity: amplification only proceeds when all six regions of the target are correctly recognized, so a chance match at one or two sites is not enough to give a signal.

Methods for Detection

The amplification and detection of DNA can be accomplished in a single step. The procedure of LAMP technology can be completed by incubating the mixture of sample primers, DNA polymerase having strand displacement activity, and substrate at a constant temperature of 60- 65°C. Samples are loaded into instruments and run all at the same time for real-time results. DNA will be highly amplified within 15 to 60 minutes.

For real-time detection, turbidity can be measured by a photometer with an incubation function, without any added detection reagent, because amplification produces insoluble magnesium pyrophosphate that makes the solution cloudy. Color-based detection uses a pH indicator such as phenol red. Amplification releases hydrogen ions and lowers the pH, so a positive reaction turns from pink to yellow, while a negative reaction stays pink. This visible change can be read by eye without any instrument.

Other than visual detection by colorimeter, the amplified products are detected by various methods. Such methods are lateral flow, agarose gel detection, and Real-time fluorescence detection using intercalators or probes.

Benefits of LAMP

LAMP is preferable to PCR and other diagnostic methods because of the following reasons:

• LAMP-based assays have been highly sensitive and specific with real-time detection.
• The process is rapid, often completed within 60 minutes.
• It uses a DNA polymerase with strong strand displacement activity, such as Bst polymerase, which is what allows the reaction to run at a single temperature.
• The protocol is simple, without any complicated procedure and different preparatory steps as in many PCR systems; therefore, it requires low-cost equipment; and the entire process is run under constant temperature.
• LAMP is less sensitive to inhibitory substances present in biological samples.
• Different primers designed to hybridize six or eight parts of the target sequence of DNA make the process highly specific because amplification occurs only when the primers properly recognize all the regions within a target DNA. And thus, DNA produced is considerably higher.
• LAMP can also be used for RNA templates by adding reverse transcriptase.

Applications of LAMP

Since its development, Lamp has been used for detecting various pathogens, including viruses, bacteria, and protozoans. In many countries, LAMP is recommended for routine identification and surveillance of pathogens. LAMP is suitable for areas with no infrastructures and other facilities available, but the population is exposed to many dangerous infectious diseases. LAMP has been applied in different diagnoses, which are summarized in the following points:

• Many food-borne diseases caused by Salmonella, Legionella, Listeria, VTEC, Norovirus, Campylobacter, etc., can be detected using the LAMP technique, which avoids lengthy culture methods.
• Detection of food pathogens by using LAMP techniques ensures food safety and quality. Traditional plating of food samples to a series of selective or non-selective media fails sometimes.
• The technique is also applicable for corona and influenza viruses. Rapid Colorimetric LAMP Assay Kit for colorimetric detection of SARS-CoV-2 can be used to analyze novel coronavirus causing COVID -19.
• HIV, HPV, Cryptosporidium oocysts, Vibrio cholerae, and other pathogens are also detected using the LAMP method with high sensitivity.
• LAMP has been applied to detect Plasmodium species in blood or fecal samples and their differentiation into species, such as Plasmodium falciparum, P. ovale, P. vivax, and P. malariae. Thick and thin smears used for parasites may give false-negative results, most commonly during low parasitemia.
• A LAMP assay is available for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. It is sensitive enough to detect very low numbers of bacteria, including in sputum samples.
• LAMP techniques are used to distinguish between organisms differing by only a single nucleotide polymorphism to identify closely sub-genotypes, hence identifying two easily confused species.
• Animal species identification of meat samples can employ the LAMP technique to know the wrong labeling of meat products
• Food contamination and plant pathology can be detected by using LAMP techniques.
• LAMP can also be applied for GMO screening and detection applied in plants
• LAMP techniques help screen toxic adulterants and admixtures in herbal products.

Limitations of LAMP

Although the LAMP technique is reliable and rapid, it has certain limitations:

• Difficulties in designing LAMP primers may occur, even though the software is available for designing LAMP primers.
• The high sensitivity and specificity may sometimes result in false-positive amplification, which can occur due to cross-contamination, mainly caused by aerosols in the assay. Therefore, great attention is necessary while handling biological samples to avoid aerosols formation, and sterilization must be maintained. DNA extraction must be performed carefully and quickly under sterile conditions to prevent contamination and DNA degradation.
• There may be difficulty in quantification.

Conclusion

There is the greatest need for simple and field-friendly diagnostic tools that can be readily adapted in poorly resourced laboratories to treat and control diseases effectively. At present, LAMP is a relevant alternative DNA-based amplification platform that is rapid, affordable, and accurate. Hence, it can be a better option for both developed and developing countries, where it can be routinely employed for sensitive and specific detection of pathogens.

How to Remember

Why it needs no thermocycler: the enzyme does the unzipping. PCR heats the DNA to pull the strands apart. LAMP does not, because Bst polymerase shoves the old strand aside as it copies (strand displacement). No heating step means no cycling machine. One temperature, start to finish.

Why the loops form: the inner primers are two-in-one. FIP and BIP each carry two joined sequences (an F2 and an F1c). That builds a strand with two complementary regions on it, so the strand folds back on itself into a loop. Loop at each end gives the dumbbell. The dumbbell is what lets the reaction self-cycle. The "loop" in the name is literally this.

Six primers, six regions: that is where the specificity comes from. More regions that must all match means far less chance of a false signal. This is why LAMP is even more specific than PCR, which uses only two.

LAMP vs PCR in one line. PCR for the equipped lab and for quantifying. LAMP for the field: faster, cheaper, no thermocycler, read by eye.

Key exam facts in one table

Where Students Get Confused

"Why does LAMP not need a thermocycler when PCR does?" Because the two methods separate DNA strands differently. PCR uses heat (about 95 °C) to pull the strands apart, so it must cycle the temperature up and down. LAMP uses Bst polymerase, which displaces the old strand as it copies, so the strands are separated by the enzyme itself. No heating step is needed, and the whole reaction runs at one temperature.

"Why does LAMP need so many primers?" The four to six primers recognize six to eight separate regions of the target. Two of the primers (FIP and BIP) are inner primers built from two joined segments each, and these are what make the DNA fold into loops. More recognition regions also make LAMP very specific: amplification only happens when all the regions match, so accidental matches do not give a false result.

"What actually makes the loops?" The inner primers. Because FIP carries both an F2 and an F1c segment, the strand it helps build contains two complementary regions (F1 and F1c) on the same strand. Complementary regions on one strand pair with each other, so the strand folds back into a loop. The same happens at the other end with BIP, giving the dumbbell shape.

"Is LAMP more or less specific than PCR?" More specific. PCR uses two primer sites; LAMP uses six or more. Because every region must be recognized for amplification to proceed, LAMP is less likely to give a false positive from a chance match. Its main downside is not specificity but the risk of contamination-driven false positives and the difficulty of quantifying the result.

"What is RT-LAMP, and how is it different?" RT-LAMP is LAMP for RNA targets. A reverse transcriptase first copies the RNA into DNA, then the normal LAMP reaction proceeds. It is how LAMP detects RNA viruses such as SARS-CoV-2. Some Bst enzymes have reverse transcriptase activity built in, so a separate enzyme is not always needed.

References

1. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), e63. https://doi.org/10.1093/nar/28.12.e63
2. Mori Y., Notomi T. (2009). Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy, 15(2), 62–69. https://doi.org/10.1007/s10156-009-0669-9
3. Chen X., Zhang J., Pan M., Qin Y., Zhao H., Qin P., et al. (2021). Loop-mediated isothermal amplification (LAMP) assays targeting 18S ribosomal RNA genes for identifying P. vivax and P. ovale, and mitochondrial DNA for detecting the genus Plasmodium. Parasites & Vectors, 14(1), 278. https://doi.org/10.1186/s13071-021-04764-9
4. Foo P.C., Nurul Najian A.B., Muhamad N.A., Ahamad M., Mohamed M., Yean Yean C., Lim B.H. (2020). Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis of LAMP, conventional PCR, nested PCR, and real-time PCR based on Entamoeba histolytica DNA. BMC Biotechnology, 20(1), 34. https://doi.org/10.1186/s12896-020-00629-8
5. Li J., Xiong C., Liu Y., Liang J., Zhou X. (2016). Loop-mediated isothermal amplification (LAMP): emergence as an alternative technology for herbal medicine identification. Frontiers in Plant Science, 7, 1956. https://doi.org/10.3389/fpls.2016.01956
6. New England Biolabs. Loop-Mediated Isothermal Amplification (LAMP). https://www.neb.com/en-us/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp