Fall/Winter 2020 expressa scientific updateions IN THIS ISSUE Feature article 2 Loop-mediated isothermal amplification (LAMP) – a fast-growing technology with a wide range of applications Supporting 5 COVID-19 research Application Note 9 Facilitating Detection of SARS-CoV-2 Directly from Patient Samples: Precursor Studies with RT-qPCR and Colorimetric RT-LAMP Reagents Reboot your Bench & 10 Accelerate your Research Consider these suggestions as you prepare for your return and benefit from faster workflows and kits Fastest RT-qPCR Kits 12 around! Test LUNA Universal Kits and experience the performance FEATURE ARTICLE ~ Loop-mediated isothermal amplification (LAMP) – a fast-growing technology with a wide range of applications By Joanne Gibson, Ph.D., New England Biolabs, Inc. Polymerase Chain Reaction – PCR The polymerase chain reaction (PCR) is an first heat-stable polymerase, was introduced. Prior It requires equipment such as a thermocycler and indispensable tool in many molecular biology to this discovery, a heat-labile polymerase was gel electrophoresis or real-time instrumentation, laboratories, and has been transforming research laboriously added at the start of every cycle because which require an electricity source. These are readily and medical diagnoses for decades. Over the years, it was destroyed by the high temperatures required available in well-equipped laboratories – along this technology has seen many advances. In the for the denaturation step. Scientists could now add with the knowledge required to design and carry early days before thermocyclers became available, polymerase for the entire amplification at the begin- out a PCR. However, in some instances there are researchers sat diligently by multiple water baths ning of the experiment. Reaction setup became even time-sensitive, or location-specific aspects to a study set at different temperatures, timer in hand, ready more user-friendly with the introduction of hot start that make transport of samples to a laboratory the to swap the tubes from one water bath to the next polymerases, in which dissociation from an inhibitor limiting factor in acquiring prompt, accurate results. for each temperature-specific step – denaturation, is required for activity, allowing for increased In these cases, the need for a simpler form of nucleic annealing and extension. Before the use of heated specificity and room temperature reaction set up. acid amplification became apparent – not in place thermocycler lids, mineral oil was layered on top of traditional PCR, but as an option for use in a Now, PCR is much more efficient and a mainstream of the reaction mixture to prevent evaporation. broader range of settings. laboratory technique for a wide range of applications. Then, Taq DNA Polymerase (NEB #M0273), the Loop-Mediated Isothermal Amplification – LAMP In response to this need, researchers developed a new One of the advantages of LAMP is its broad utility. Figure 1: method of nucleic acid amplification: loop-mediated LAMP reaction progress can be tracked in real time 3´ F3c F2c F1c B1 B2 B3 5´ isothermal amplification (LAMP) (1). In a nutshell, by adding a fluorescent dye that binds to dsDNA as 5´ F3 F2 3´ LAMP is a powerful method for amplifying a target it amplifies. For high throughput applications, LAMP F1c Forward internal sequence in a single tube, at a single temperature using can be incorporated into automated workflows, where 5´ primer (FIP) a variety of simple detection methods. endpoint detection is measured via an absorbance plate 5´ F1c F2 F1 B1c B2c B3c 3´ reader (2). It can also be adapted for performance out- 3´ B2 B3 5´ LAMP uses 4 to 6 primers that recognize 6 to 8 B1c side of a traditional lab, where analysis “in the field”, Backward internal regions of the target DNA or RNA sequence (Figure 1). primer (BIP) 5´ The design of two of the primers intentionally results requires a simplified output that shows amplification success. Various detection methods have been tested, 3´ F1 F2c F1c B1 B2 B1c 5´ in self-hybridizing loop structures that form a dumb- including visualizing the precipitation of magnesium bell, providing numerous sites for synthesis initiation. pyrophosphate or the color change of a metal-sensitive 3´ Bst DNA Polymerase, Large Fragment (NEB #M0275), F1c B1 F2 indicator, but both of these methods are difficult to B2 which is active at elevated temperatures and ideal for F2c F1 3´ 5´ B1c detect by eye and require approximately 60 minutes this application, displaces downstream DNA that is F1c before detection is possible. FIP encountered during strand synthesis. These features 5´ op of LAMP – the hyper priming of the target sequence, NEB scientists developed a solution to this – a very Lo B coupled with the use of a strand-displacing DNA visible, pH-based colorimetric detection (3). This 3´ F1c B1 F2 B2 Polymerase – lead to exponential amplification at a rate method utilizes a pink-to-yellow color change that is F2c F1 3´ 5´ B1c that can be detected by a variety of methods in approx- visible to the naked eye, resulting from a change in pH. F1c FIP imately 30 minutes. NEB-engineered versions of the During amplification, a proton is released with every 5´ Bst enzyme have expanded LAMP utility by reducing dNTP that is incorporated into the growing DNA Loop F inhibition by common sample contaminants and strand; in the presence of a lightly buffered solution, 3´ dUTP. Increased dUTP tolerance enabled carryover this leads to a decrease in pH of 2-3 units, which is F1 B1c F2 B2 F1c 5´ 3´ B1 B2c prevention, once common only in PCR-based tech- attributed to the large amount of DNA made in LAMP B1c niques, to be adapted to LAMP. Additionally, robust reactions. Incorporation of a pH indicator directly BIP 5´ RNA-based amplification can be accomplished by the into the reaction mixture enables visual detection of simple addition of the NEB WarmStart RTx enzyme, amplification. This eliminates the need for specialized Exponential Amplication which is also included in NEB LAMP master mixes. detection methods, such as gel electrophoresis, which Therefore, amplification of RNA or DNA substrates is not practical in remote settings where point-of-care Loop-mediated isothermal amplification (LAMP) uses 4-6 primers can be performed outside the walls of a laboratory; in diagnostics might be desirable or for high-throughput recognizing 6-8 distinct regions of target DNA. A strand-displacing fact, the entire reaction takes place at 65°C and could analysis in field studies. As a result, LAMP lends itself DNA polymerase initiates synthesis and 2 of the primers form loop be carried out in a cup of hot water. well to a variety of applications. structures to facilitate subsequent rounds of amplification. 2 passing it to the next human they bite. The injected the wMel strain of Wolbachia from fruit flies mosquito (Aedes aegypti), is one of these arboviral into Aedes aegypti eggs and the Wolbachia-infected Colorimetric LAMP disease-disseminating organisms. The World Health mosquitoes were released in Northern Australia on in the field Organization estimates that 2.5 billion people live a controlled schedule. Within a few months, close in dengue transmission areas; it is one of the top ten to 100% of mosquitoes contained Wolbachia, an dengue global health threats and the most rapidly spreading infection rate that has been retained years later (5). mosquito-borne disease. The WMP adapted LAMP for field analysis and Interestingly, 40-60% of all the different species compared it with the established TaqMan qPCR Wolbachia of insects worldwide (including butterflies and assay. LAMP primers were designed to detect the dragonflies) contain a maternally-transmitted, wsp gene from two Wolbachia strains. Using the endosymbiotic Gram-negative bacterium called WarmStart Colorimetric LAMP 2X Master Mix Wolbachia pipientis in their reproductive cells; Aedes (NEB #M1800), primers and target DNA, they aegypti mosquitoes are one of the few insects that do found that in just 30 minutes at 65°C, a simple color not normally carry Wolbachia. Once a virus change can reliably determine if the mosquitoes are (e.g., dengue) enters cells, the presence of Wolbachia infected with Wolbachia: pink = negative (mosquito in those cells prevents viral growth by slowing does not contain Wolbachia DNA), yellow = positive There is no better example of just how beneficial replication and rapidly degrading viral RNA (4), (mosquito contains Wolbachia DNA) (6). preventing downstream infections in humans. amplification in the field is than in the work Screening mosquitoes in the field using colorimetric conducted by the World Mosquito Program (WMP) This potential biocontrol strategy was recognized LAMP is inexpensive compared to the TaqMan (worldmosquitoprogram.org), whose research aims by the WMP and researchers embarked on a qPCR assay, and it avoids the lengthy process to eradicate vector-borne illnesses. massive effort to stop transmission by disseminating of transporting samples back to a specialized Dengue, chikungunya and Zika viruses are trans- Wolbachia-containing mosquitoes in dengue- laboratory for analysis. Results are in real time, and mitted by arthropods that acquire the diseases by endemic countries in Central and South America this improves accuracy regarding the geographical feeding on infected human blood and sub sequently and into Asia and the Pacific regions. Researchers localization of the Wolbachia-containing mosquitoes. The field of space biology is still in its infancy but ends of chromosomes that support chromosomal an ever-expanding list of biological experiments is stability by protecting them from degradation; Colorimetric LAMP being conducted on the International Space Station abnormal telomere shortening is associated with in space (ISS) each year, some with the help of students, human disease. The colorimetric LAMP assays that who design these experiments as part of the Genes were designed to assess telomere dynamics were in Space contest (www.genesinspace.com).