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Accurate, Fast, Simple – The master of LAMP Detection

2022-07-22

 
Due to the outbreak of coronavirus disease (COVID-19) in 2019, qRT-PCR has been the standard method for virus detection, but the rapid mutation and strong infectivity of COVID-19 was unexpected. Because of the infection speed and virus detection need to be operated by healthcare professional, finally these reasons caused medical burden. In order to reduce the current situation, Emergency Used Authorization (EUA) has been distributed around the world which allowed to the development and production of antigen rapid test, helping the public can self-screening to reduce medical resources. It takes about 15 minutes to obtain the result, but the methodology is based on immunoassay with low sensitivity unlike PCR which can amplify signal. In addition, most of the antigen rapid test detect Nucleocapsid Protein, therefore there is a risk of cross-reactivity with similar antigenic structures, resulting in a higher chance of false negative/false positive results.

How can we find a method that is accuracy, high sensitivity and rapid detection? Let me introduce to you: "Loop-mediated isothermal amplification, LAMP"!


What is LAMP?
PCR is the most commonly used nucleic acid amplification technique at present. Although it has high sensitivity and specificity, but there are still limitations because PCR is more complicated to operate and requires higher technical requirements for instruments and technician, so it is not suitable for rapid diagnosis or field investigation. However, in 2000, Dr. Notomi and his team [1] published a new genetic diagnostic technique in the journal Nucleic Acids Res, a technology of isothermal amplification, LAMP (Loop-mediated isothermal amplification).
       
The LAMP reaction requires two pairs of primers, as shown in Figure 1, the outer primer (F3, B3) and the inner primer (FIP, BIP). Compared to the normal PCR method, which requires only one pair of primers, where the primers must be designed for the specific region of the target sequence to ensure the specificity of the target. In addition to need Bst DNA polymerase, which has 5'→ 3' DNA polymerase activity and strong strand displacement activity and activate at a constant temperature of 60-65 degrees. According to M.M.Parida et al. proved that using LAMP can amplification DNA into 109 in one hour. [2]P.R. Sahoo and his team also found that if want to increase the amplification efficiency, a third pair of primers, loop primer (LF, LB), can be added to accelerate the DNA synthesis by annealing at F1/F2 and B1/B2 regions.[3]

 


Figure 1: LAMP application flow chart

The principle of LAMP
When LAMP successfully amplifies DNA, it will release pyrophosphate which will produce water-insoluble magnesium pyrophosphate white precipitates with the magnesium ions (Mg2+) in the reaction. Therefore, LAMP can not only be observed by the naked eye, but also can be used with fluorescent reagent to detect the DNA amplification. The reaction time is less than an hour. Table 1 shows the differences between LAMP and PCR.

Table 1: Comparison of LAMP and PCR methodologies
 
However, LAMP still has its shortcomings, (1) primer specificity highly recommends, (2) the amplified DNA can only be used for distinguish and cannot be used for subsequent experimental applications (such as cloning), (3) due to the high sensitivity of LAMP, it is easy to cause false-positive results due to aerosol formation.
 

Application of LAMP
LAMP has been widely used and many commercial products have been developed for detecting different diseases. In the past decade, food was contaminated by bacteria such as Salmonella, Campylobacter, and E. coli which causing vomiting, diarrhea, and gastroenteritis. Therefore, LAMP can be used to detect food-infection. In developed countries, the LAMP detection is very suitable where medical resources are insufficient. Because the lack of medical resources, often accompanied by epidemic diseases, such as tuberculosis and HIV.[4]
 
In these 2-3 years, due to the outbreak of COVID-19, many researchers and institutions have developed prototype of LAMP assays for the diagnosis of COVID-19. Lamb et al. first develops an assay that can effectively detect COVID-19 within 30 minutes and in their study, they tested different disease sources to demonstrate the high specificity of LAMP. The Penn-RAMP method published by El-Tholoth et al. that both colorimetric and fluorescence assays achieved good result and the performance of purified targets was ten times more sensitive than conventional RT-PCR. Anahtar MN et al. used chemical RNase inactivation by TCEP/EDTA and heat-mediated cleavage to increase LAMP 30% sensitivity, while also reducing viral infectivity and decrease human-risk. Broughton JP et al. combined RT-LAMP with a CRISPR-Cas12 diagnostic method using COVID-19 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), based on CRISPR analysis considering the absence of virus-related genes in human cells, to improve detection efficiency. [5]
 
I'm sure you have more understand of LAMP and exciting to develop your own assay reagents using this technology. But you may consider about spending time and money to prepare the materials in the early stage. You don't have to worry about this! TOOLS also provides LAMP technology related products, the operation is simple and time-saving, just need observe the color change by your eyes and you can quickly detect viruses and micro pathogen!
 
What are you waiting for? Click the link below and know more information!
TOOLSee Colorimetric RT-LAMP kit (ST-L01)
TOOLSee Saliva Colorimetric RT-LAMP kit (ST-SL01)

 
Reference:
  1. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000 Jun 15;28(12):E63.
  2. M.M.Parida,S.Sannarangaiah,P.K.Dash,P.V.L.Rao,K.Morita,Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases, Rev.Med.Virol.(2008),doi:10.1002/rmv.593.
  3. P.R.Sahoo,K.Sethy,S.Mohapatra,D.Panda, Loop mediated isothermal amplification: an innovative gene amplification technique for animal diseases, Vet.World9(2016)465–469,doi:10.14202/vetworld.2016.465-469.
  4. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009 Apr;15(2):62-9. doi: 10.1007/s10156-009-0669-9. Epub 2009 Apr 25. PMID: 19396514; PMCID: PMC7087713.
  5. Chaouch M. Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2. Rev Med Virol. 2021 Nov;31(6):e2215. doi: 10.1002/rmv.2215. Epub 2021 Jan 21. PMID: 33476080; PMCID: PMC7995099.
  6. Collins RA, Ko LS, So KL, Ellis T, Lau LT, Yu AC. Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA. J Virol Methods. 2002 May 16;103(2):213-25. doi: 10.1016/s0166-0934(02)00034-4. PMID: 12008015.
  7. Böhmer A, Schildgen V, Lüsebrink J, Ziegler S, Tillmann RL, Kleines M, Schildgen O. Novel application for isothermal nucleic acid sequence-based amplification (NASBA). J Virol Methods. 2009 Jun;158(1-2):199-201. doi: 10.1016/j.jviromet.2009.02.010. Epub 2009 Feb 14. PMID: 19428591.

 
 
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