DNA amplification is an indispensable technique used in molecular biology laboratories, with polymerase chain reaction (PCR) being the most popular method. The core principle of PCR is the use of high heat to separate strands of DNA, followed by cooling to a lower temperature to allow a DNA polymerase enzyme to copy the separated strands. The reaction cycles through these steps many times - denaturation, annealing and extension – and it requires precise and rapid temperature change, enabled by sophisticated laboratory instrumentation. However, in some instances where DNA amplification is required, there are time-sensitive or location-specific aspects to a study that make transport of samples to a laboratory undesirable. Therefore, the need for a simpler method of nucleic acid amplification became apparent – not in place of traditional PCR, but as an option for use in a broader range of settings.
Loop-mediated isothermal amplification (LAMP)
In response to this need, researchers developed loop-mediated isothermal amplification (LAMP), a powerful method for amplifying a target sequence of DNA or RNA in a single tube, at a single temperature, compatible with various simple detection methods.
To recognize the regions of the target sequence, LAMP uses four to six primers, two of which are designed to create self-hybridizing loop structures that form a dumbbell and create numerous sites for synthesis initiation. The high specificity and sensitivity of the primers lead to exponential amplification that can be detected in less than 30 minutes.
With the development of engineered strand-displacing DNA polymerases and reverse transcriptases, LAMP can be performed outside the walls of a laboratory; in fact, the entire reaction takes place at 65°C and can be carried out in a cup of hot water. However, field-based analysis also requires a simplified method of detection. For this reason, scientists developed a viable solution – a pH-based colorimetric detection, which utilizes a pink-to-yellow color change that is visible to the naked eye.
During amplification, a proton is released with every deoxynucleoside triphosphate added into the growing DNA strand, leading to a decrease in the pH. Incorporating a pH indicator into the reaction mixture enables colorimetric detection, thereby eliminating the need for specialized detection methods, which are not practical in point-of-care and field settings.
The power of LAMP during a pandemic
During a global pandemic, rapid, widespread testing for the infectious agent is critical. Reverse transcription quantitative PCR (RT-qPCR) is the standard for diagnosing acute infections, and testing is typically conducted in authorized labs that can perform high-complexity tests. However, during the ongoing COVID-19 pandemic, the scientific community saw the need to develop faster, alternative testing approaches beyond the traditional clinical laboratory.
The minimal instrument requirements and portability of colorimetric LAMP have huge potential as a disease-screening method because of the low cost, rapid results and ease-of-use in a broad range of settings. During the initial phases of the pandemic, scientists demonstrated the ability of a colorimetric LAMP assay to quickly detect RNA from the SARS-CoV-2 virus from the respiratory swabs of confirmed infections from patients in Wuhan, China (Figure 1). After that initial report, numerous groups have taken advantage of LAMP to expand COVID testing worldwide. Use has been demonstrated in high-throughput testing labs and simple, mobile collection directly from saliva or swab samples. As we continue to need more and broader testing, these approaches utilizing LAMP can supplement the traditional PCR testing paradigm and aid in the fight against COVID.
Figure 1: SARS-CoV-2 detection from COVID-19 patient samples in Wuhan, China. Samples testing positive (1-6) or negative (7) with commercial RT-qPCR tests were assayed using colorimetric LAMP assay with primer set targeting ORF1a (A) and GeneN (B). Yellow indicates a positive detection after 30 min incubation, and pink a negative reaction with results compared to the negative control (N). B, Blank control without template. P, samples containing a plasmid used as positive control for qPCR. Credit: Zhang et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric lamp. medRxiv. 2020. doi:10.1101/2020.02.26.20028373
The future potential of LAMP as a field-based tool
The value of colorimetric LAMP lies in its simple and inexpensive application in tropical disease settings, which are not typically PCR-friendly. For example, dengue is one of the top-ten global health threats and the most rapidly spreading mosquito-borne disease. More than 2.5 billion people live in dengue-transmission subtropical regions, according to the World Health Organization.
Dengue cannot replicate in mosquitoes that carry the endosymbiont Wolbachia pipientis, so the World Mosquito Program (WMP) embarked on a massive effort to stop transmission of dengue (and other vector-borne viruses) by disseminating Wolbachia-containing mosquitoes. However, scientists had to first identify the mosquitoes that carried Wolbachia. They could do this using colorimetric LAMP technology – within 30 minutes and through a simple color change.
Screening mosquitoes in the field using colorimetric LAMP is inexpensive compared to the typical qPCR assay. It avoids the lengthy process of transporting samples back to a specialized laboratory for analysis. Results are in real-time, and this improves accuracy regarding the geographical localization of the Wolbachia-containing mosquitoes.
These field-friendly advantages can also be utilized for agriculture. Grapevine red blotch virus (GRBV) is a recently identified virus that infects grapevines across North America, resulting in a reduction in yield and quality. Characteristic leaf changes (i.e., red blotches) can be easily observed. Yet, visual detection alone is not reliable, which is why diagnostic testing is required. Sample testing using a colorimetric LAMP assay can take place onsite, which reduces the time and cost of screening GRBV when compared to traditional methods.
LAMP in space . . . and beyond
The first PCR assay was conducted in space in 2016 aboard the International Space Station (ISS). However, with the goal of extended space travel, there is a need for astronauts to analyze reactions without having to send samples back to Earth. That happened in March 2017, when colorimetric LAMP experiments, designed by a high school student as part of the Genes in Space contest, were conducted on the ISS to study changes in telomere dynamics while in outer space.
As part of the experiment, two assays were prepared on Earth and then carried out on the ISS: one using traditional PCR and the other using colorimetric LAMP. While the traditional PCR assays had to be transported back to Earth for analysis, the colorimetric LAMP assays were assessed on the ISS as they required only visual inspection to indicate successful amplification – photographing the tubes against a white piece of paper highlighted the simplicity of the methodology and its suitability for even one of the most extreme settings.
These examples illustrate the ability of colorimetric LAMP to bring nucleic acid amplification to new settings with a simple and inexpensive application. Colorimetric LAMP opens up opportunities for human health and medical diagnosis that, until recently, have not been possible due to the specialized requirements of traditional PCR. Any study environment where samples currently need to be transported from the field to a specialized lab is an opportunity to develop a colorimetric LAMP assay.