Colorimetric LAMP for molecular detection
Colorimetric LAMP is a simple, intuitive, inexpensive, sensitive and specific* tool for the detection of pathogens and various genetic markers at point-of-need. In theory, LAMP should be highly specific. However, due to its nonspecific amplification of LAMP primers in the absence of a target template, its specificity is greatly compromised, and it often produces false positives. Our scientists have discovered an inhibitor that can block nonspecific LAMP amplification without affecting specific amplification. It allows us to develop a new colorimetric LAMP reagent, DirectLAMP, which we believe could finally realize the potential of colorimetric LAMP as a molecular detection and diagnostic tool for widespread uses at resource-limited settings.
How Colorimetric LAMP works
Loop-mediated isothermal amplification (LAMP) is a technique that can be used to amplify and detect specific DNA or RNA sequences with high sensitivity and specificity under isothermal conditions, typically at a constant temperature between 63°C and 65°C.
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LAMP works by using a set of four to six specially designed primers that recognize distinct regions of the target DNA sequence. The primers initiate a strand displacement reaction that creates a looped structure with a stem and loop that rapidly amplifies the target DNA sequence in a highly specific manner. The reaction results in the production of large amounts of DNA and other by-products that can be detected using various detection methods, including the simple and cost-effective colorimetric LAMP.
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In colorimetric LAMP, a colorimetric indicator is added to the reaction mixture, which changes color when the DNA is amplified. The indicator usually consists of a pH-sensitive dye, such as phenol red, that changes color from purple to yellow as the pH of the reaction mixture decreases due to the production of hydrogen ion during the amplification process.
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The color change can be visually detected with the naked eye and recorded with a cell phone camera without the need for expensive equipment or specialized training. This makes colorimetric LAMP ideal for point-of-care diagnostics, field-based testing, testing in remote areas with limited access to laboratory facilities, and even for at-home self-testing.
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Colorimetric LAMP has been used for the detection of various pathogens, including viruses, bacteria, and parasites, as well as genetic disorders and mutations. It is a promising tool for the rapid and specific detection of infectious diseases and has the potential to improve global health outcomes.
The principle of colorimetric LAMP. Colorimetric LAMP reagent contains a sensitive pH indicator, phenol red. Phenol red's color changes from purple to yellow as hydrogen ion concentration increases during DNA amplification.
Inhibition of primer amplification by Boltii Diagnostics' nonspecific LAMP amplification inhibitor. Reactions 11-14 contained no inhibitor and Reactions 15-18 contained the inhibitor. Reactions 11, 13, 15, 17 were no-template controls (solid lines) and Reactions 12, 14, 16, 18 contained 200 copies of SARS-CoV-2 virions (dashed lines). Magenta intensity in the reaction was monitored as an indication of DNA amplification for 120 min. Without the inhibitor, DNA was amplified irrespective of the presence of the virions (11-14). With the inhibitor, DNA was not amplified in the absence of the virions (15, 17) but only amplified in the presence of the virion (16, 18).
Nonspecific LAMP amplification in the absence of a template
LAMP has demonstrated great potential as a diagnostic tool. However, its widespread use has been limited by the nonspecific amplification of primer sequences in the absence of a target sequence. This nonspecific amplification reduces the specificity of LAMP and makes it unreliable as a diagnostic tool.
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One of the biggest challenges for LAMP users is the presence of primer amplification in their no-template controls. It can impede their research progresses significantly. They may start out thinking it a cross-contamination and spend weeks trying to identify possible sources of contamination. As amplification in NTC persists, they may begin to suspect it a nonspecific primer amplification and send the LAMP products out for DNA sequencing, which would confirm their suspicions. Next, they would likely redesign and order more primer sets to be tested in LAMP experiments. However, none of the new primer sets could avoid the nonspecific primer amplification. For many users, this often means the end of their LAMP projects.
Scientists have attempted to address this problem using two approaches. The first approach involves checking for the presence of the amplified target sequence in the amplification products using DNA sequencing, target-specific oligonucleotide probes, or CRISPR detection. While this approach has improved the specificity of LAMP detection, it has also increased the complexity and cost of the detection process. Additionally, this approach is not applicable to the simple and intuitive colorimetric LAMP detection method.
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The second approach involves adding various additives to LAMP reactions to inhibit or delay nonspecific primer amplification. Little progress had been made in this area until our recent breakthrough in discovering a substance that can inhibit nonspecific LAMP amplification while retaining specific LAMP amplification. This discovery has allowed us to develop a novel colorimetric LAMP reagent, DirectLAMP, which has the potential to eliminate nonspecific LAMP amplification and prevent false positives.
DirectLAMP inhibits nonspecific LAMP amplification
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DirectLAMP is a colorimetric LAMP Master Mix that contains our proprietary nonspecific LAMP amplification inhibitor. The inhibitor prevents nonspecific LAMP amplification while allows for specific LAMP amplification. This makes DirectLAMP a reliable tool that can prevent false positives in colorimetric LAMP tests.
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Using DirectLAMP is easy. Simply add one volume (1 uL) of your primers and one volume (1 uL) of sample to 10 volumes (10 uL) of DirectLAMP Master Mix and start the LAMP reaction. After two hours of incubation at 63-65°C, the reaction reaches equilibrium with maximal DNA and hydrogen ion production.
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The test results are simple to interpret. If the color of the no-template control (NTC) reaction and the test reaction remain the same (purple/red/pink), there is no LAMP amplification, and the test is negative. If the NTC reaction remains purple/red/pink while the test reaction changes to orange or yellow, there is LAMP amplification, and the test is positive. If the NTC turns orange or yellow, the test is invalid, indicating possible cross-contamination with the target sequence.
LAMP reaction color changed in the absence and presence of Boltii Diagnostics' nonspecific LAMP amplification inhibitor. Reactions 11-14 contained no inhibitor and Reactions 15-18 contained the inhibitor. Reactions 11, 13, 15, 17 were no-template controls and Reactions 12, 14, 16, 18 contained 200 copies of SARS-CoV-2 virions. Without the inhibitor, DNA was amplified (reaction color turned yellow in 11-14) irrespective of the presence of the virions. With the inhibitor, DNA was not amplified in the absence of the virions (reaction color remained red in 15, 17) but amplified in the presence of the virions (reaction color turned yellow in 16, 18). There is no need to stop the reaction by 95°C heating at the end of the reaction. There won't be any amplification in the NTCs even after they are left at room temperature for days.