Monitoring tick and mosquito infections - using the LAMP technology for infectious disease diagnostics and surveillance
This blog post describes the development and perspectives of the LAMP (Loop-mediated Isothermal Amplification) technology as point-of-care diagnostic methods, for which it took a pandemic to find its stride.
LAMP constitutes a novel opportunity for low-resource testing in remote areas of life-threatening diseases, agricultural diagnostics such as testing crops for infection; and field surveillance programs involving disease vector insects and ticks.
The Carlow lab at NEB has used LAMP technology for infectious disease diagnostics and surveillance for almost a decade. They have developed LAMP tests to detect insect and tick-borne diseases such as Eastern Equine Encephalitis, anaplasmosis, Lyme Disease, and babesiosis, and various Neglected Tropical Diseases (NTDs), such as lymphatic filariasis (elephantiasis) and onchocerciasis (River Blindness).
Key advantages of LAMP technology compared to PCR:
Requires only basic hot block (65°C), no expensive equipment such as a real-time thermocycler
Requires very little technical lab expertise, no threshold (Ct) value and amplification curve interpretations
Quick turnaround time (30 minutes)
Visible-to-the-eye read-out, no specialized fluorescence detection or electrophoresis equipment needed
Reduced contamination risk, as colour change output is visualized without opening tubes
Ability to lyophilize LAMP reagents, no need for refrigeration at the testing site
Figure 1: The simplified LAMP workflow requires no sophisticated equipment and can be carried out without an
electrical source, with results in 30 minutes that are visible to the naked eye. LAMP is ideal for screening and
diagnosing NTDs in low-resource settings, agriculture, tick and mosquito monitoring programs, and even at-home
testing for infections.
The technology behind the LAMP method
LAMP uses four core and two additional “loop” primers, producing a short dumbbell structure that forms the basis for exponential amplification. The need of a thermocycler and high temperatures to denature the DNA strands in the PCR reaction, is replaced by incorporating a strand-displacing DNA polymerase, which extends the primer while displacing the duplex. A more thorough explanation of LAMP is displayed in this video.
LAMP makes a LOT of DNA quickly and within 30 minutes. There are various outputs available. When the Carlow Lab started exploring LAMP, the readout was turbidity-based. This wasn’t easy to interpret because it involves a change in color from purple to shades of blue. Thus, researchers at NEB developed another colorimetric readout of the LAMP results based on a pH change that is much easier to detect by the eye.
Figure 2: Simple, fast, and clear read-out of the LAMP reaction. The visual detection is based on the production of protons and subsequent drop in pH, occurring from the extensive DNA polymerase activity in a LAMP reaction, effecting a change in solution color from pink to yellow.
Development of LAMP as a diagnostic tool
LAMP is well-suited for diagnostics; however, it doesn’t replace PCR for some downstream applications such as cloning. It can be used for sequencing, but generally, a rapid yes/no diagnostic answer is sufficient for the intended application.
Scientists at NEB have worked to bring a suite of LAMP expertise and tools up to the molecular diagnostic standards of PCR. By 2015, they had engineered enhanced LAMP enzymes such as Bst 2.0, controlled their activity for high-throughput and room-temperature setup with novel WarmStart® aptamers, and figured out how to prevent carryover contamination with dUTP and a new thermolabile UDG. They developed a method for LAMP multiplexing to detect multiple targets, optimized the pH-based colorimetric detection method described above, and launched master mixes and glycerol-free enzymes – an essential requirement for lyophilization.
Fast forward to 2020, and it was quickly realized that LAMP had some application in testing for SARS-CoV-2. Supply chain shortages and labs struggling to keep up with testing needs highlighted the benefit of a diagnostic test that could be applied with minimal cost, materials, and molecular expertise. This brought LAMP to the forefront as many researchers pivoted to address testing needs. NEB scientists established an in-house CLIA-certified SARS-CoV-2 LAMP assay using saliva with minimal sample prep for NEB employees.
At the same time the Carlow Lab was spearheading the CLIA lab LAMP-based testing, they were also involved in various LAMP-based collaborations, namely optimizing a colorimetric Onchocerciasis diagnostic LAMP assay in Cameroon and developing a colorimetric RT-LAMP assay for the detection of Eastern Equine Encephalitis (EEE) virus in vector mosquitoes. Read more below.
NEB's LAMP products:
|Product name||Product number||Size(s)||Order|
|Bst 3.0 DNA Polymerase||M0374S/M||1.600/ 8.000 units||Order|
|Bst 2.0 DNA Polymerase||M0537S/M||1.600/ 8.000 units||Order|
|Bst 2.0 Warm Start DNA Polymerase||M0538S/M||1.600/ 8.000 units||Order|
|Bst DNA Polymerase||M0275S/M||1.600/ 8.000 units||Order|
|WarmStart RTx Reverse Transcriptase||M0380S/L||50 / 250 rxns||Order|
|WarmStart LAMP Kit (DNA & RNA)||E1700S/L||100 / 500 rxns||Order|
|WarmStart Fluorescent LAMP/RT-LAMP Kit with UDG||E1708S/L||100 / 500 rxns||Order|
|SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit||E2019S||96 rxns||Order|
Until October 31st 2022, we offer you 20% discount on these products!
Colorimetric LAMP improves the accessibility of EEEV testing in the U.S.
EEE is an arbovirus transmitted by mosquitoes. Although rare, an infection can cause brain inflammation and is often fatal. It has an enzootic cycle between mosquitoes (Culiseta melanura is the primary mosquito vector) and passerine birds.
While RT-PCR is the current gold standard for EEE surveillance, it is often not implemented because of access and funding limitations. Mosquito control programs around the U.S. typically don’t have much funding, and it varies from state to state and even between counties within a state. The cost of sending samples to a specialized lab can stifle a minimally funded surveillance program.
In a study published earlier this year (Maddison et al., 2022) based on a collaboration between the Carlow lab and Tom Unnasch’s lab at the University of South Florida, a LAMP assay was developed to detect EEE virus. The initial primer design process is critical for a successful assay, and NEB scientists and software engineers have streamlined the process with a LAMP Primer Design web tool. The method was demonstrated to be a highly accurate and tremendously robust assay alternative to RT-PCR.
Figure 3: Analytical sensitivity of RT-LAMP for EEEV. (A) Colorimetric RT-LAMP assays targeting nsP3, E1, or 6K, were performed using serially diluted viral RNA (PFU/ml) as a template. The colour change from pink to yellow indicates a positive result. (B) The percentage of samples that tested positive at each serial dilution (PFU/ml) of viral RNA using LAMP primers sets for nsP3 (dark green bar), E1 (medium green bar), or 6K (light green bar) are shown (calculation based on biological replicates of each PFU concentration run in duplicate with each primer set).
The LAMP assay developed in this collaboration used three highly conserved EEE virus biomarkers (nsP3, E1, and 6K) that target different regions of the viral genome. When RT-PCR was compared with LAMP, 100% agreement was observed between the results from the quantitative (Ct) values obtained with RT-PCR and the end-point colour change (pink to yellow). It should be noted, however, that RT-PCR can detect down to approximately 1/10 of a virus particle, but a single infectious mosquito carries about one million virus particles. So, in that sense, all three RT-LAMP biomarkers were comparable in terms of being able to detect a single infectious mosquito.
All-in-all, this is a cost-effective, simple, fast, and accurate assay that can be implemented at the level of local testing programs; not just for EEE mosquito monitoring, but for many surveillance and diagnostic programs that would benefit from nucleic acid amplification in a simplified format.
Marianne Møller Brorson