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Vol 25, No. 03, March 2021   |   Issue PDF view/purchase
FEATURES
The Silence of the LAMP
As the demand for accurate molecular diagnostics increases, it is time for loop-mediated isothermal amplification assay, or LAMP, to make noise.
Mark B. Carascal

In a typical peak day for COVD-19 swab testing, Sheila, a medical technologist in a major tertiary hospital in Metro Manila, is expecting five hundred swab samples for processing. With her meticulous eyes and trained hands, processing of samples for COVID-19 testing is already like reciting the lyrics of her favourite song. Sheila, like most medical technologists in city testing centres, relies on her technical skills, boosted by machines for molecular testing—but what about the under-equipped and low-resource testing centres with no access to the needed skills and equipment? Is there a way to test for infections with an alternative molecular technique that has uncompromised accuracy, requires only few technical resources, and costs less? Loop-mediated isothermal amplification, a technology introduced to the scientific community for more than two decades now, seems to be a perfect fit as an alternative platform.

Loop-mediated isothermal amplification, commonly referred to as LAMP, was first described in 2000 by Japanese researchers, led by Dr. Tsugunori Notomi, in an attempt to address the limitations of existing nucleic acid amplification tests (NAATs). Similar to real-time polymerase chain reaction, the current gold standard for COVID-19 testing, LAMP is an assay that detects the presence of a target genetic sequence in a sample. As with any molecular diagnostic tests, LAMP uses primers to find and amplify its target until detectable amounts of products are produced. However, unlike the typical NAATs that use only two primers, LAMP uses four to six primers to detect its target. Moreover, LAMP forms loop structures to further hasten the amplification process, hence the name “loop-mediated.”

“LAMP is one of those innovative and cost-effective amplification platforms for nucleic acids detection,” described by Dr Raul Destura, an award-winning inventor, molecular biologist, and infectious disease specialist in the Philippines. With more than fifteen years of experience in developing molecular diagnostic kits for infectious diseases, he considers LAMP as among the most promising platforms. His affirmation to the promises of LAMP is attributable to its various desirable characteristics as a molecular diagnostic assay.

Turn the LAMP on

Among the common descriptions of LAMP in the scientific literature, two qualities stand-out: LAMP is rapid and sensitive. LAMP is rapid since a typical assay would only require a reaction time of no more than sixty minutes. One study even reported a complete reaction time of only fifteen minutes to amplify the target sequence. Meanwhile, it is sensitive because it can detect the target sequence even at the concentration of as low as ten copies in a single reaction. Just think of it as looking for a needle in a haystack—only with LAMP, you are more likely to find the needle.

Aside from the common advantages described in the scientific literature, LAMP also offers practical advantages. “[LAMP] is an ideal molecular diagnostic method, especially in under-equipped conditions, because it does not require costly or specialized devices such as thermal cyclers,” described by Dr Windell Rivera, an academician of the National Academy of Science and Technology, and one of the Philippine’s most renowned parasitologists. According to him, unlike most NAATs, LAMP does not require costly or specialized devices like thermal cyclers because the amplification of targets occurs at a constant temperature, hence the name “isothermal.” A typical LAMP reaction can amplify target sequences at the usual temperatures of 60 to 65 Degrees Celsius. Because of this property, one can perform LAMP assays by using ordinary heat blocks or constant temperature water baths.

Another practical advantage of LAMP is the ease of detecting its end-products. Typically, scientists and technologists detect products of NAATs through sophisticated processes like gel electrophoresis or fluorescence detection. LAMP products can also be detected through similar methods or through simpler visualization methods like turbidity measurement, colorimetric assessments, or fluorescence through UV or LED illumination. These additional methods make it possible to use the naked eye where no instrumentation is needed in determining whether a LAMP reaction is successful or not.

Although LAMP offers several advantages over the other NAATs in the current molecular diagnostic pipelines, the method is not perfect. Over the years, scientists are still working hard to improve the workflow for LAMP assays.

LAMP buster

One of the major disadvantages of using LAMP for diagnostic applications lies on its own strength—its high sensitivity. “The high sensitivity of the LAMP system makes it susceptible to false positives because of carry-over or cross-contamination,” Dr Rivera recalled based from his laboratory experience. This is corroborated by Dr Destura who highlighted that the skills of the technicians doing the assay are also important. “One of the challenges in the field is when the laboratory personnel have limited skills [in] micro-pipetting; [this limitation increases the] risk of cross-contamination [especially] when they are not careful,” he mentioned. Micro-pipetting is the process of transferring very small amounts of liquid from one container to another, and is a very important skill in any molecular biology workflow such as LAMP. Meanwhile, cross-contamination refers to the phenomenon when LAMP products unintentionally contaminate other setups leading to false positive results.

Another disadvantage of LAMP is its limited versatility in terms of post-amplification processing. Since the products of LAMP reactions are contiguous and of different sizes (hence the typical ladder-like visualized products), post-amplification processing like purification, cloning, and sequencing become almost impossible. In addition, primer design and extended multiplexing capabilities are also persisting challenges. To address these limitations, researchers developed various primer design software for LAMP, and introduced innovative techniques like the Detection of Amplification by Release of Quenching (DARQ), and Quenching of Unincorporated Amplification Signal Reporters (QUASR).

The limitations of using LAMP are often seen as the reasons why it is not a very popular NAAT compared to PCR (Polymerase Chain Reaction). The silence of the LAMP in both the scientific community and applied industries may be apparent, but many researchers and innovators are still seeing its potential.

LAMP in Asia

Despite the current limitations of using LAMP, many innovators in Asia used this platform in establishing adoptable and low-cost technologies for use in the laboratories or even in the field. “[LAMP] assay has been used as a point-of-care diagnostics for some pathogenic diseases in humans and other animals,” said Dr Mary Beth Maningas, an award-winning scientist, and developer of LAMP technologies for the aquaculture. Her team, composed of young scientists and entrepreneurs (G. Buela, V. Samora, C. Caipang, A. Nicolasora, B. Maralit, J. Vergel), is part of the University of Santo Tomas’ ongoing venture, the Aqua Gen Tech Company (AGTC), which aims to bring diagnostic products for use of the aquaculture farmers. Their flagship technology is called Juan Amplification (JAMP) Alert kit, a LAMP-based kit for detecting White Spot Syndrome Virus (WSSV) infection in shrimps. WSSV causes the fatal and contagious white spot disease in shrimps; hence, its early detection ensures control of the infection, and promotes more bountiful and healthy harvest.

Meanwhile, Dr Destura is the innovator of a world-renowned dengue diagnostic kit based on LAMP: the Biotek-M™ Dengue Aqua kit, manufactured by the first spin-off company of the University of the Philippines- National Institutes of Health, the Manila HealthTek, Inc. As one of the most significant tropical infectious diseases in Southeast Asia, the detection of dengue infection is critical. However, the current platforms in diagnosing dengue remain challenging particularly for those who do not have access to high-end technologies. Hence, Dr Destura’s inspiration in making the kit is clear-cut: “The inspiration is really access—access to molecular tools, [and] molecular amplification technology at a cost that is affordable [to] the general Philippine population.”

Dr. Rivera, who serves as the principal investigator of the University of the Philippines-Pathogen-Host-Environment Interaction Research Laboratory (PHEIRL), was involved in the development of published LAMP assays in detecting parasitic protozoans Entamoeba histolytica (cause of amoebiasis) and Trichomonas vaginalis (cause of trichomoniasis). Currently, his laboratory is also developing a LAMP technology for the detection of Salmonella enterica, the cause of an intestinal tract infection, salmonellosis, attributable to contaminated meat products. His laboratory believes that the development of molecular diagnostic tools that can be applied and adopted in the actual field and industries will help foster more accurate quality control systems. In the long run, this can also make LAMP a more familiar NAAT to everyone.

For the rest of the Asia, diagnostic products based on LAMP are also available, primarily for the detection of viruses such as Japanese encephalitis virus, West Nile virus, human papillomavirus, and Zika virus; in parasites such as Plasmodium falciparum (cause of malaria); and in bacterial pathogens such as Mycobacterium tuberculosis (cause of tuberculosis). With the advent of the COVID-19 pandemic, a question then arises: can LAMP be used in the development of COVID-19 diagnostics?

Silent no more

“LAMP is a promising tool for COVID-19 diagnostics. Since SARS-CoV-2 is an RNA virus, the causative agent of COVID-19, reverse transcription-LAMP (RT-LAMP) is the right approach. This method is simple and highly specific because it uses more sets of primers in contrast to PCR,” Dr Rivera mentioned. Dr Maningas stated that there are currently a number of published studies in China and USA that used LAMP as a molecular diagnostic test in detecting SARS-CoV-2. At the moment, the team of Dr Destura is also developing a LAMP-based kit for COVID-19. Although clinical validation is still necessary before implementation of LAMP-based diagnostic assays, its future and applicability in the clinical setting are very promising. Currently, a number of LAMP-based technologies have already been given the emergency use authorization by the US FDA (US Food and Drug Administration) for the detection of COVID-19.

As for disease diagnostics in general, LAMP remains to be a candidate for high-demand utilization. “With the on-going research and development for LAMP technologies, I am envisioning LAMP point-of-care [technologies] for several diseases, be it in the aquaculture, livestock, and even more for human diseases,” said Dr Maningas. “After all, alternative technologies that work similar to the current gold standard NAATs, but at a lower cost, and at a more adoptable workflow, are very important options in the diagnostics of the future,” Dr Destura added.

As Sheila processes the remaining batch of swab samples for COVID-19 testing, another batch of samples arrived from the neighbouring city that depends on Sheila’s laboratory for testing. Can you imagine if the diagnostic technology for testing COVID-19 does not require specialized laboratory equipment for processing? Can you imagine if all cities, and even provinces, have the power to conduct their own testing with a diagnostic technology that they can readily adopt? We hope that the potential of LAMP technologies will be heard, and its capabilities be used for more applications in the different fields of diagnostics in the future. For now, let’s hope that Sheila can still manage to process the increasing amounts of samples, with the continuously increasing number of COVID-19 suspects in Metro Manila.

References:

  • Ball CS, Light YK, Koh CY, Wheeler SS, Coffey LL, Meagher R. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses. Analytical Chemistry. 2016; 88(7): 3562–3568. doi:10.1021/acs.analchem.5b04054
  • Karthik K, Rathore R, Thomas P, et al. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination. MethodsX. 2014;1:137-143. Published 2014 Aug 27. doi:10.1016/j.mex.2014.08.009
  • Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16(3):223-9. doi: 10.1006/mcpr.2002.0415
  • Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi:10.1093/nar/28.12.e63
  • Quoc NB, Phuong NDN, Chau NNB, Linh DTP. Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood. Heliyon. 2018;4(3):e00561. Published 2018 Mar 6. doi:10.1016/j.heliyon.2018.e00561
  • Saharan P, Khatri P, Dingolia S, Duhan JS, Gahlawat SK. Rapid Detection of Viruses Using Loop-Mediated Isothermal Amplification (LAMP): A Review. Biotechnology: Prospects and Applications. 2013;287-306. Published 2013 Oct 4. doi:10.1007/978-81-322-1683-4_21
  • Sahoo PR, Sethy K, Mohapatra S, Panda D. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases. Vet World. 2016;9(5):465-469. doi:10.14202/vetworld.2016.465-469
  • Tanner NA, Zhang Y, Evans TC Jr. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. Biotechniques. 2012 Aug;53(2):81-9. doi: 10.2144/0000113902

About the Author

Mark Carascal is a scientist, science illustrator, and science communication advocate. He is a Registered Microbiologist and Science Research Specialist at the Clinical and Translational Research Institute of The Medical City, Philippines. He is also a graduate student at the Institute of Biology, University of the Philippines Diliman. He is currently involved in the development and validation of loop-mediated isothermal amplification assays for the detection of multidrug-resistance genes in human pathogens

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