Loop-Mediated Isothermal Amplification (LAMP)

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.

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.

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).

DNA amplification in LAMP can be divided into three steps illustrated in the figure (figure source: intechopen.com). 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 and primes the polymerase by adding complementary nitrogen bases in the 3′ to 5′ 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, backward inner primer (BIP) binds to the newly made complementary strand at the B2c region, synthesizing complementary DNA in the 3′ to 5′ direction.
• Now, the backward outer primer, F3, 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 contains two complementary regions, F1 and F1c, and B1 and B1c; these regions bind to each other, making a dumbbell-shaped structure with two loops.

Cycling amplification step

• Here, the looped structure forms the basis for the LAMP auto cycling process.
• Then, FIP and BIP primers bind at F2c and B2c, respectively, self-priming of F1-F1c and B1-B2c occurs, and it continues cycling resulting in 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.

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 the detection, real-time turbidity measurement can be performed by photometer with incubation function, which does not need any detection reagents. The color change is observed as yellow for a positive result, whereas orange or pink for a negative one. The strong color change is based on a pH change during the reaction.

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 different DNA polymerases, tolerant to high strand displacement activity (e.g., Bst polymerase).
• 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.
• LAMP assay is available for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. It can detect a single copy of bacteria in specimen and media samples in sputum m TB in sputum sample has been found.
• 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.

References

1. 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 species and mitochondrial DNA for detecting the genus Plasmodium. Parasites &Amp; Vectors, 14(1). https://doi.org/10.1186/s13071-021-04764-9
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. Biolabs, N. (2022). International.neb.com. Retrieved 2 June 2022, from https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
4. 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. https://doi.org/10.3389/fpls.2016.01956
5. Foo, P., Nurul Najian, A., Muhamad, N., Ahamad, M., Mohamed, M., Yean Yean, C., & Lim, B. (2020). Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample. BMC Biotechnology, 20(1). https://doi.org/10.1186/s12896-020-00629-8