NEB
EXPRESSIONS
A scientific update
Issue II • 2020
Loop-mediated isothermal amplification (LAMP) – an emerging technology with a wide range of applications
Feature Article
Facilitating detection of SARS-CoV-2 directly from patient samples
Technical Note
– –
Product Highlights
Learn how researchers are using their expertise to advance coronavirus detection methods
COVID-19 Researcher Spotlight
NEB wants to help you quickly get back to the important science you do
Reboot Your Bench
Supporting the development of COVID-19 diagnostics and vaccines GMP-grade reagents for RNA synthesis provide the tools to take you from template to transcript
Australia & New Zealand New England Biolabs (Australia) PTY Telephone: 1800 934 218 (AU) Telephone: 0800 437 209 (NZ) www.nebiolabs.com.au www.nebiolabs.co.nz
Canada New England Biolabs, Ltd. Toll Free: 1-800-387-1095 www.neb.ca
China New England Biolabs (Beijing), Ltd. Telephone: 010-82378265/82378266 www.neb-china.com
USA New England Biolabs, Inc. Telephone: (978) 927-5054 Toll Free: (U.S. Orders) 1-800-632-5227 Toll Free: (U.S. Tech) 1-800-632-7799 www.neb.com
France New England Biolabs France Telephone : 0800 100 632 www.neb-online.fr
Germany & Austria New England Biolabs GmbH Free Call 0800/246 5227 (Germany) Free Call 00800/246 52277 (Austria) www.neb-online.de
Japan New England Biolabs Japan, Inc. Telephone: +81 (0)3 5669 6191 www.nebj.jp
Singapore New England Biolabs Pte. Ltd. Telephone +65 638 59623 www.neb.sg
United Kingdom New England Biolabs (UK), Ltd. Call Free 0800 318486 www.neb.uk.com
View contacts in other countries
Loop-mediated isothermal amplification (LAMP) –
The polymerase chain reaction (PCR) is an indispensable tool in many molecular biology laboratories, and has been transforming research and medical diagnoses for decades. Over the years, this technology has seen many advances. In the early days before thermocyclers became available, researchers sat diligently by multiple water baths set at different temperatures, timer in hand, ready to swap the tubes from one water bath to the next for each temperature-specific step – denaturation, annealing and extension. Before the use of heated thermocycler lids, mineral oil was layered on top of the reaction mixture to prevent evaporation. Then, Taq DNA Polymerase (NEB #M0273), the first heat-stable polymerase, was introduced. Prior to this discovery, a heat-labile polymerase was laboriously added at the start of every cycle because it was destroyed by the high temperatures required for the denaturation step. Scientists could now add polymerase for the entire amplification at the beginning of the experiment. Reaction setup became even more user-friendly with the introduction of hot start polymerases, in which dissociation from an inhibitor is required for activity, allowing for increased specificity and room temperature reaction set up.
By Joanne Gibson, Ph.D., New England Biolabs
Polymerase Chain Reaction – PCR
Loop-Mediated Isothermal Amplification – LAMP
In response to this need, researchers developed a new method of nucleic acid amplification: loop-mediated isothermal amplification (LAMP) (1). In a nutshell, LAMP is a powerful method for amplifying a target sequence in a single tube, at a single temperature using a variety of simple detection methods. LAMP uses 4 to 6 primers that recognize 6 to 8 regions of the target DNA or RNA sequence (see video below). The design of two of the primers intentionally results in self-hybridizing loop structures that form a dumbbell, providing numerous sites for synthesis initiation. Bst DNA Polymerase, Large Fragment (NEB #M0275), which is active at elevated temperatures and ideal for this application, displaces downstream DNA that is encountered during strand synthesis. These features of LAMP – the hyper priming of the target sequence, coupled with the use of a strand-displacing DNA Polymerase – lead to exponential amplification at a rate that can be detected by a variety of methods in approximately 30 minutes. NEB-engineered versions of the Bst enzyme have expanded LAMP utility by reducing inhibition by common sample contaminants and dUTP. Increased dUTP tolerance has enabled carryover prevention, once common only in PCR-based techniques, to be adapted to LAMP. Additionally, robust RNA-based amplification can be accomplished by the simple addition of the NEB WarmStart RTx enzyme (NEB #M0380), which is also included in NEB LAMP master mixes. Therefore, amplification of RNA or DNA substrates can be performed outside the walls of a laboratory; in fact, the entire reaction takes place at 65°C and could be carried out in a cup of hot water.
One of the advantages of LAMP is its broad utility. LAMP reaction progress can be tracked in real time by adding a fluorescent dye that binds to dsDNA as it amplifies. For high throughput applications, LAMP can be incorporated into automated workflows, where endpoint detection is measured via an absorbance plate reader (2). It can also be adapted for performance outside of a traditional lab, where analysis “in the field”, requires a simplified output that shows amplification success. Various detection methods have been tested, including visualizing the precipitation of magnesium pyrophosphate or the color change of a metal-sensitive indicator, but both of these methods are difficult to detect by eye and require approximately 60 minutes before detection is possible. NEB scientists developed a solution to this – a very visible, pH-based colorimetric detection (3). This method utilizes a pink-to-yellow color change that is visible to the naked eye, resulting from a change in pH. During amplification, a proton is released with every dNTP that is incorporated into the growing DNA strand; in the presence of a lightly buffered solution, this leads to a decrease in pH of 2-3 units, which is attributed to the large amount of DNA made in LAMP reactions. Incorporation of a pH indicator directly into the reaction mixture enables visual detection of amplification. This eliminates the need for specialized detection methods, such as gel electrophoresis, which is not practical in remote settings where point-of-care diagnostics might be desirable or for high-throughput analysis in field studies. As a result, LAMP lends itself well to a variety of applications.
a fast-growing technology with a wide range of applications
About PCR
About LAMP
There is no better example of just how beneficial amplification in the field is than in the work conducted by the World Mosquito Program (WMP) (worldmosquitoprogram.org), whose research aims to eradicate vector-borne illnesses. Dengue, chikungunya and Zika viruses are transmitted by arthropods that acquire the diseases by feeding on infected human blood and subsequently passing it to the next human they bite. The mosquito (Aedes aegypti), is one of these arboviral disease-disseminating organisms. The World Health Organization estimates that 2.5 billion people live in dengue transmission areas; it is one of the top ten global health threats and the most rapidly spreading mosquito-borne disease. Interestingly, 40-60% of all the different species of insects worldwide (including butterflies and dragonflies) contain a maternally-transmitted, endosymbiotic Gram-negative bacterium called Wolbachia pipientis in their reproductive cells; Aedes aegypti mosquitoes are one of the few insects that do not normally carry Wolbachia. Once a virus (e.g., dengue) enters cells, the presence of Wolbachia in those cells prevents viral growth by slowing replication and rapidly degrading viral RNA (4), preventing downstream infections in humans. This potential biocontrol strategy was recognized by the WMP and researchers embarked on a massive effort to stop transmission by disseminating Wolbachia-containing mosquitoes in dengue-endemic countries in Central and South America and into Asia and the Pacific regions. Researchers injected the wMel strain of Wolbachia from fruit flies into Aedes aegypti eggs and the Wolbachia-infected mosquitoes were released in Northern Australia on a controlled schedule. Within a few months, close to 100% of mosquitoes contained Wolbachia, an infection rate that has been retained years later (5). The WMP adapted LAMP for field analysis and compared it with the established TaqMan® qPCR assay. LAMP primers were designed to detect the wsp gene from two Wolbachia strains. Using the WarmStart® Colorimetric LAMP 2X Master Mix (NEB #M1800), primers and target DNA, they found that in just 30 minutes at 65°C, a simple color change can reliably determine if the mosquitoes are infected with Wolbachia: pink = negative (mosquito does not contain Wolbachia DNA), yellow = positive (mosquito contains Wolbachia DNA) (6). Screening mosquitoes in the field using colorimetric LAMP is inexpensive compared to the TaqMan qPCR assay, and 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.
LAMP in the Field
LAMP in Space
LAMP During a Pandemic
LAMP in Agriculture
Overview of LAMP
The field of space biology is still in its infancy but an ever-expanding list of biological experiments is being conducted on the International Space Station (ISS) each year, some with the help of students, who design these experiments as part of the Genes in Space contest. In early experiments, reactions to be conducted on the ISS were typically prepared on Earth before being executed in space, and then transported back to Earth where they could be analyzed and interpreted. In this way, the first PCR was conducted in space in 2016 aboard the ISS (7). But with the goal of extended space travel there remained a need to enable astronauts to analyze reactions that could be conducted on station without having to send samples back down to Earth. In March 2017, colorimetric LAMP experiments were conducted on the ISS to detect a specific repetitive telomeric DNA sequence and observe the colorimetric results (8). Telomeres are DNA-protein structures at the ends of chromosomes that support chromosomal stability by protecting them from degradation; abnormal telomere shortening is associated with human disease. The colorimetric LAMP assays that were designed to assess telomere dynamics were prepared on the ground but after amplification on station using a portable miniPCR™ instrument, the results (a simple pink to yellow color change enabled by the WarmStart Colorimetric LAMP Master Mix) were easily visualized, indicating successful amplification. Traditional PCR using both Taq and Q5® DNA polymerases (NEB #M0494) were run in parallel and all reactions were compared to those run on Earth to demonstrate the study’s success. Analysis of the LAMP results on the ISS was made possible by the fact that colorimetric LAMP required only visual inspection – 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.
During a global pandemic, rapid, widespread testing of the infectious agent is critical – not just for testing symptomatic cases, but also as a screening method as people return to work, and to generally monitor infection levels in a community. The SARS-CoV-2 virus causes symptoms that resemble the flu, so molecular diagnosis is essential for confirmation of infection and epidemiological monitoring. RT-qPCR is the standard for the diagnosis of acute infections from upper and lower respiratory tract specimens, and testing is typically conducted in authorized labs that can perform high complexity tests. However, the scientific community is collaborating to develop faster, alternative approaches to detect this pathogen. Scientists at NEB demonstrated the ability of a colorimetric LAMP assay to detect RNA from the SARS-CoV-2 virus that causes COVID-19 (9). The assay was evaluated using samples from respiratory swabs from confirmed infections from patients in Wuhan, China (Figure 1) where isothermal amplification of SARS-CoV-2 RNA leads to a color change from pink to yellow (Figure 2). Additional studies have examined colorimetric LAMP in comparison to commercial RT-qPCR tests (10). LAMP can be used with samples that do not require RNA isolation (11), including those using human saliva as a sample input (12). NEB recently released a research-use only (RUO) product based on this simple technique, the SARS CoV-2 Rapid Colorimetric LAMP Assay Kit (NEB #E2019). Colorimetric LAMP has huge potential as a disease screening method during a pandemic because of the low cost, rapid results and ease of use in a broad range of settings. The quick sample preparation and portability of this assay represent the next-generation method of enabling our customers point-of-need diagnostics that will aid in disease prevention or containment and lead to improved outcomes.
The widespread use of colorimetric LAMP doesn’t end there. Due to its sensitivity and low cost (under a dollar per sample) it is also being applied in agriculture. For example, Grapevine red blotch virus (GRBV) is spread by the three-cornered alfalfa leafhopper and it affects the viticulture industry in North America. It causes a reduction in both yield and quality, and while characteristic leaf changes are observed, they are not reliable and so diagnostic testing is required. The sample collection is as simple as inserting a sterile pipette into the leaf petiole and then incubating the tip in sterile water. Sample testing using a colorimetric LAMP assay takes place onsite (13). Compare this with the time and cost of traditional methods for screening for GRBV – sample collection, transport to a specialist lab, a 2-hour DNA extraction method and PCR analysis. Additionally, the sensitivity of the LAMP assay was reported to be 2 orders of magnitude lower than traditional PCR and qPCR methods. The value of colorimetric LAMP lies in its simple and inexpensive application in study settings that have not previously been PCR-friendly. 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 whereby samples currently need to be transported from the field to a specialized lab is an opportunity for the development of a colorimetric LAMP assay.
Now, PCR is much more efficient and a mainstream laboratory technique for a wide range of applications. It requires equipment such as a thermocycler and gel electrophoresis or real-time instrumentation, which require an electricity source. These are readily available in well-equipped laboratories – along with the knowledge required to design and carry out a PCR. However, in some instances there are time-sensitive, or location-specific aspects to a study that make transport of samples to a laboratory the limiting factor in acquiring prompt, accurate results. In these cases, the need for a simpler form of nucleic acid amplification became apparent – not in place of traditional PCR, but as an option for use in a broader range of settings.
LAMP
in agriculture
in space
during a pandemic
in the field
Figure 2. Rapid, simple, color-based detection of SARS-CoV-2 RNA: The SARS-CoV-2 Rapid Colorimetric LAMP Assay, targeting the nucleocapsid (N) and envelope (E) genes, was carried out using the indicated controls and either positive sample (human total RNA + synthetic SARS-CoV-2 RNA) or negative sample (human total RNA alone). Valid results for Non-Template Control (NTC, pink), Positive Control (PC, yellow) and Internal Control (IC, yellow) are shown. In the positive test sample, isothermal amplification of SARS-CoV-2 RNA leads to a color change from pink to yellow.
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.
Loop Mediated Isothermal Amplification (LAMP) Tutorial
References: 1. Notomi, T. et al. (2000) Nucl. Acids. Res., 28:12, e63. PMID: 10871386. 2. Zhang, Y. et al. (2020) BioTechniques. DOI 10.2144/btn-2020-0078. 3. Tanner, N.A. et al. (2015) BioTechniques, 58(2):59-68. PMID: 25652028.
References: 4. Thomas, S. et al. (2018) PLoS Pathogens, 14 (3): e1006879. PMID: 29494679. 5. Ryan, P.A. et al. (2020) Gates Open Res. 3:1547. 6. Gonçalves, D.D.S. et al. (2019) Parasit Vectors. 2019;12(1):404.
References: 7. Boguraev, A.S. et al. (2017) NPJ Microgravity 3:26 8. Rubenfien, J. et al. (2020) FASEB BioAdvances, 2:160-165. PMID: 32161905.
References: 9. Zhang, Y. et al. (2020) medRxiv, 2020.02.26.20028373. 10. Anahtar, M.N. et al. (2020) medRxiv, 2020.05.12.20095638. 11. Dao Thi, V.L. et al. (2020) medRxiv, 2020.05.05.20092288. 12. Lalli, M.A. et al. (2020) medRxiv, 2020.05.07.20093542.
References: 13. Romero, J.L.R. et al. (2019) Arch Virol., 164(5):1453-1457.
Facilitating Detection of SARS-CoV-2 Directly from Patient Samples
Precursor Studies with RT-qPCR and Colorimetric RT-LAMP Reagents
by Andrew N. Gray, Ph.D., Guoping Ren, Ph.D., Yinhua Zhang, Ph,D., Nathan Tanner, Ph.D. and Nicole M. Nichols, Ph.D., New England Biolabs, Inc.
Introduction
Materials
Results
Discussion
Summary
INTRODUCTION This Application Note is presented in the context of the COVID-19 global pandemic caused by the SARS-CoV-2 coronavirus. At the time of writing, over two million COVID-19 cases with over 150,000 deaths have been confirmed globally (1). The number of individuals infected with SARS-CoV-2 is likely much higher, however, owing in part to limited testing. It has been widely acknowledged that broader testing is critically needed. In the United States, most diagnostics approved to date under FDA Emergency Use Authorization (EUA) have employed RT-qPCR for hydrolysis probe-based (e.g., TaqMan®) detection of viral targets in RNA purified from patient samples. The inclusion of an up-front RNA purification step, however, adds to protocol time and cost per sample, and overall can reduce testing throughput. In addition, the scale of demand for testing materials has led to supply shortages for some RNA purification reagents. Diagnostics that are capable of robust detection directly from patient samples would therefore be highly advantageous, potentially enabling testing that is faster, cheaper, higher-throughput, and better positioned to scale for anticipated demand.
To help accelerate diagnostics development efforts, here we demonstrate detection of synthetic SARS-CoV-2 viral RNA targets using NEB’s RT-qPCR and colorimetric RT-LAMP reagents. The colorimetric RT-LAMP work is a follow-up on a recent publication by researchers at NEB, in collaboration with researchers at the Wuhan Institute of Virology in China, demonstrating colorimetric RT-LAMP detection of SARS-CoV-2 viral RNA purified from patient samples (2). Further, we evaluate two requisite features for direct detection from patient samples: tolerance to Universal Transport Medium and resistance to inhibition and/or RNA degradation in the presence of human cell lysates.
At the time of writing, over two million COVID-19 cases with over 150,000 deaths have been confirmed globally.
Back to top
MATERIALS • Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) • Luna Universal Probe One-Step RT-qPCR Kit (ROX-free) (NEB #E3007)* • Luna Cell Ready Probe One-Step RT-qPCR Kit (NEB #E3031) • Luna Cell Ready Lysis Module (NEB #E3032) • LunaScript RT SuperMix Kit (NEB #E3010) • WarmStart Colorimetric LAMP 2X Master Mix (NEB #M1800)
* Note: This is a new product released to meet recent increased demand. Performance is equivalent to NEB #E3006 for real-time instruments that do not use ROX signal-based reference dye normalization.
Direct Detection of SARS-CoV-2 RNA Targets in Cell Lysates Direct detection of SARS-CoV-2 in patient samples will require that reagents be resistant to inhibition by cell lysates, and that viral RNA be protected from degradation by cellular RNases prior to detection. To assess both these factors, we lysed cells in the presence of synthetic SARS-CoV-2 RNA (vRNA) and then assessed detection and quantitation via both one-step RT-qPCR and colorimetric RT-LAMP, using purified vRNA alone as a control. Cell lysis was carried out using the Luna Cell Ready Lysis Module, which includes an RNA Protection Reagent to prevent RNA degradation. It should be noted that the synthetic vRNA used here should only be regarded as a partial proxy for targets within the full-length, capsid-enclosed viral RNA genome (see Discussion Section). Using the Luna Universal Probe One-Step RT-qPCR Kit, SARS-CoV-2 vRNA targets were detected with high sensitivity (consistent detection at approximately 50 copies per reaction; lower titers not yet tested), in both the absence and presence of cell lysates (Figures 3A-C). Additionally, linear quantitation was observed over a broad range of vRNA titers (50 to 500,000 copies, efficiency = 93.8% for Orf1a quantitation, 99.1% for N-gene quantitation) (Figures 3A,B). Cell lysates ranged in density from 0.2 to 2,000 cell equivalents per RT-qPCR reaction, and equivalent tolerance was observed over this range.
DISCUSSION The data presented here is intended to serve as a starting point, which we hope will enable further studies and test development. Additional questions remain, and development of direct diagnostics will ultimately require testing with COVID-19 patient samples. NP swab samples contain not only UTM, nasal epithelial cells (and/or lysed cell contents) and viral particles, but also mucus and other nasal mucosal secretions; direct testing of samples will be required to determine whether these additional factors affect reagent performance and/or viral RNA stability during testing workflows. Additionally, while naked synthetic RNA was used in our testing as a proxy, most viral RNA in patient samples is sequestered in proteinaceous capsids within enveloped virion particles. This protects viral RNA from RNases secreted by nasal mucosa, and thus helps prevent RNA degradation prior to testing; however, the virion envelope and enclosed capsid must ultimately be disrupted to enable viral RNA detection. Despite these challenges, a recent study demonstrated direct detection of SARS-CoV-2 RNA in patient samples using Luna One-Step RT-qPCR reagents (3). While a drop in sensitivity was observed for direct detection (Cq = 23) compared to quantitation from purified RNA for the same samples (Cq = 18.7), this was still far above apparent detection limits for the assay, suggesting that direct detection could be viable even at much lower viral titers than those of the patient samples tested. The observed Cq delay may result from incomplete viral RNA release from encapsidation, reagent inhibition, and/or viral RNA degradation; evaluating and mitigating these factors may further improve assay performance.
While RT-qPCR has been the dominant form of COVID-19 diagnostic testing thus far, a test employing colorimetric RT-LAMP could provide several critical advantages. First, it allows broad accessibility as a field or point-of-care diagnostic, as it only requires incubation at a single reaction temperature (for isothermal amplification) and employs visual detection. In contrast, RT-qPCR requires more expensive real-time PCR instrumentation to allow thermocycling and fluorescence detection. Second, RT-LAMP is rapid, with results after ≤ 30 minutes of incubation. Colorimetric RT-LAMP thus offers the potential for a simple, rapid, inexpensive and broadly deployable test allowing in-visit diagnosis in both lab and point-of-care settings, and even testing in the field at points of need to aid surveillance and prevention.NEB has and will continue to supply and support customers who are working diligently to develop better diagnostic tools for the COVID-19 virus. We hope that the work described here may contribute to these efforts.
A recent study demonstrated direct detection of SARS-CoV-2 RNA in patient samples using Luna One-Step RT-qPCR reagents
SUMMARY
• • •
World Health Organization. Coronavirus Disease 2019 https://www.who.int/emergencies/diseases/novel-coronavirus-2019 (2020). Zhang, Y. et al. Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv 2020.02.26.20028373 (2020) (preprint) doi:10.1101/2020.02 .26.20028373. Bruce, E.A. et al. RT-qPCR Detection of SARS-CoV-2 RNA from Patient Nasopharyngeal Swab using Qiagen RNEasy Kits or Directly via Omission of an RNA Extraction Step. bioRxiv 2020.03.20.001008 (2020) (preprint) doi:10.1101/2020.03.20.001008.
A-B. Simultaneous quantitation of SARS-CoV-2 N-gene (A) and human actin mRNA (B) from human total RNA (5 pg to 50 ng) spiked with synthetic SARS-CoV-2 viral RNA (50 to 500,000 copies), in the presence (darker color) vs absence (lighter color) of 5 μl UTM per reaction (25% v/v), as determined by multiplex RT-qPCR using the Luna Universal One-Step Probe RT-qPCR Kit. Synthetic viral RNA was generated by in vitro transcription of RNA corresponding a portion of N-gene from the SARS-CoV-2 genome (GenBank Accession Number MN908947). C-D. Quantitation of actin (C) or N-gene (D) by Two-Step RT-qPCR using the LunaScript RT SuperMix Kit for first-strand cDNA synthesis and Luna Universal Probe qPCR Master Mix for subsequent amplification and quantitation.
FIGURE 1: Tolerance of Luna RT-qPCR reagents to Universal Transport Medium
A. Colorimetric LAMP (left, DNA target) and RT-LAMP (right, RNA target) were carried out in the presence of UTM (Copan Diagnostics) at the indicated volumes. 10 ng human genomic DNA was used as template for detection of the BRCA gene, and 5 ng human total RNA was used for detection of β-actin mRNA. B. Amplification during Colorimetric LAMP/RT-LAMP quantitated using a fluorescent dye.
FIGURE 2: Tolerance of WarmStart Colorimetric LAMP 2X Master Mix to Universal Transport Medium
Multiplex RT-qPCR quantitation of SARS-CoV-2 N-gene (A) Orf1a (B) and human actin mRNA (C), from synthetic vRNA alone (lighter color) versus human (HeLa) cells lysed in the presence of synthetic vRNA (darker color). Lysis was conducted using the Luna Cell Ready Lysis Module, and RNA targets were quantitated using the Luna Universal Probe One-Step RT-qPCR Kit. For vRNA, input ranged from 50 to 500,000 copies per reaction. For vRNA plus lysate, cells were diluted in tandem with vRNA prior to lysis, giving 0.2 to 2,000 cell equivalents per reaction.
FIGURE 3: Direct detection and quantitation of SARS-CoV-2 RNA targets in cell lysates by One-Step RT-qPCR
WarmStart Colorimetric LAMP 2X Master Mix was used to detect SARS-CoV-2 RNA targets in cell lysates. Human (HeLa) cells were lysed in the presence of synthetic vRNA using the Luna Cell Ready Lysis Module. Lysates were then diluted 1:10, and 1 μl then added to each RT-LAMP reaction, giving 48 to 4,800 vRNA copies as indicated and 2 to 200 cell equivalents per reaction.
FIGURE 4: Direct detection of SARS-CoV-2 RNA targets in cell lysates by RT-LAMP
RT-qPCR
Colorimetric RT-LAMP
RESULTS Reagent tolerance to Universal Transport Medium (UTM) Nasopharyngeal (NP) swabs are the predominant patient sample type recommended for SARS-CoV-2 diagnostic testing and are most commonly stored in Universal Transport Medium (UTM) prior to processing. UTM tolerance is thus a critical reagent feature for direct detection of SARS-CoV-2 in patient samples. We therefore tested the effects of UTM on candidate NEB reagents for viral RNA detection, including the Luna® Universal Probe One-Step RT-qPCR Kit (for hydrolysis probe-based detection of RNA), LunaScript® RT SuperMix Kit (for first-strand cDNA synthesis in two-step RT-qPCR workflows), and WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (for rapid reverse transcription and isothermal amplification of RNA targets, with a simple visible detection). Both the Luna one-step and two-step RT-qPCR reagents were highly tolerant of UTM, with no detectible effect on detection and quantitation with 5 μl UTM per 20 μl reaction (25% v/v) (Figure 1A-D). Sensitive detection and linear quantitation were observed for both a human mRNA target (actin) from human total RNA (Figures 1A,C) and a SARS-CoV-2 viral RNA target (N-gene) from synthetic viral RNA (Figures 1B,D).
UTM at up to 25% (v/v) total reaction volume does not affect performance of either the Luna Universal Probe One-Step RT-qPCR Kit (for One-Step viral RNA detection and quantitation) or the LunaScript RT SuperMix Kit (for first-strand cDNA synthesis from viral RNA in Two-Step RT-qPCR workflows). Synthetic SARS-CoV-2 viral RNA is protected from degradation by cellular RNases during Luna Cell Ready lysis protocols, and during subsequent detection and quantitation by Luna One-Step RT-qPCR. Detection and quantitation of viral RNA targets using the Luna Universal Probe One-Step RT-qPCR Kit is achievable with cell lysates, at up to 2,000 cell equivalents per 20 μl reaction.
UTM at up to 8% (v/v) total reaction volume is tolerated for color change detection using the WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA), and at up to 20% (v/v) for amplification using alternative detection methods (e.g., fluorescence). Detection and quantitation of viral RNA targets using colorimetric RT-LAMP can be achieved with cell lysates, at up to 200 cell equivalents per 25 μl reaction. For color change detection, Luna Cell Ready lysates and other buffered lysates should be diluted prior to addition to colorimetric RT-LAMP reactions. We recommend adding 1 μl of a 1:10 dilution per 25 μl reaction as a starting point.
References 1. 2. 3.
Supporting COVID-19 Research
As the SARS-CoV-2 virus continues to impact our communities, our manufacturing and distribution teams continue to be fully operational, and we are working closely with our suppliers and distribution partners to ensure uninterrupted access to our products and technical support. NEB’s products are available for research purposes only. However, we are supplying and supporting customers who are working diligently to develop better diagnostic tools and vaccines for the SARS-CoV-2 virus, and are ready to supply additional customers with the reagents they need to validate and develop them as diagnostic tools for lab-based or point-of-care settings.
Learn more about the products available for these applications:
RNA extraction
Virus detection
Sequencing & Epidemiology
Vaccine development
Learn How NEB is Supporting COVID-19 Research
Monarch® Nucleic Acid Purification Kits
Extraction of RNA is often the first step in viral detection assays. The Monarch Total RNA Miniprep Kit can be used to successfully extract viral RNA from various samples including saliva, buccal swabs, and nasopharyngeal samples*. Extraction can be completed in under an hour with our new simplified workflow. Additionally, the Monarch RNA Cleanup Kits, which were originally developed for RNA cleanup applications, have been shown to successfully extract viral RNA from saliva and swabs when used in conjunction with the Monarch DNA/RNA Protection Reagent (NEB #T2011). This extended utility of the RNA cleanup kits enables purification of viral RNA in under 10 minutes. Both the Monarch Total RNA Miniprep Kit and RNA Cleanup Kit workflows are automatable with minor protocol modifications on the QIACube® and the KingFisher™ Flex platforms, to further facilitate efficient extraction. RNA purified with these kits is ready for downstream detection by RT-qPCR, RT-LAMP or other methods. *NEB has confirmed viral RNA extraction on simulated nasopharyngeal samples.
< Back to applications
Learn More
Protocol for Total RNA Purification from Saliva, Buccal Swabs and Nasopharyngeal Samples
Featured Monarch RNA protocol
View Protocol
Efficient and fast extraction of viral RNA from saliva, buccal swabs, and nasopharyngeal samples*
Enables sensitive detection by RT-qPCR, RT-LAMP and other methods
Automatable on the QIAcube and KingFisher Flex
Virus Detection
SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (NEB #E2019)
Luna® Universal Probe One-Step RT-qPCR Kit (NEB #E3006) & Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007)
View Citations for Isothermal Amplification, RT-qPCR and COVID-19
SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit
The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit utilizes loop-mediated isothermal amplification for use in the analysis of SARS-CoV-2, the novel coronavirus that causes COVID-19.The kit includes WarmStart Colorimetric LAMP 2X Master Mix with uracil-DNA glycosylase (UDG) and a primer mix targeting the N and E regions of the viral genome. Controls are provided to verify assay performance and include an internal control primer set and a positive control template. Guanidine hydrochloride has been shown to increase the speed and sensitivity of the RT-LAMP reaction and is also included. WarmStart Colorimetric LAMP 2X Master Mix with UDG is an optimized formulation of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx in a special low-buffer reaction solution containing a visible pH indicator for rapid and easy detection of LAMP and RT-LAMP reactions. The inclusion of dUTP and UDG in the master mix reduces the possibility of carryover contamination between reactions.This system provides a fast, clear visual detection of amplification, based on the generation of protons and subsequent drop in pH that occurs from the extensive DNA polymerase activity in a LAMP reaction. The decrease in pH produces a color change from pink to yellow. The reaction requires only a heated chamber and samples, with a run time of 30 minutes.
< Back to Virus Detection products
Reduce risk of carryover contamination, with UDG and dUTP included in the master mix
Set up reactions quickly and easily, using a simple heat source and unique WarmStart technology
Colorimetric LAMP enables simple, visual detection (pink-to-yellow) of amplification of SARS-CoV-2 nucleic acid
Assay targets N and E regions of the SARS-CoV-2 genome, for optimized sensitivity and specificity
Bring confidence to your results using the provided controls
SARS-CoV-2 genome. Arrows indicate regions targeted by assay.
Luna® Universal Probe One-Step RT-qPCR Kit and Luna Probe One-Step RT-qPCR Kit (No ROX)
The NEB Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) and Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) are optimized for real-time quantitation of target RNA sequences using hydrolysis probes. One-Step RT-qPCR provides a convenient and powerful method for RNA detection and quantitation. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, and then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via qPCR. Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5´ → 3´ exonuclease cleavage of a quenched, target-specific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle or Cq value can be determined. Cq values can be used to evaluate relative target abundance between two or more samples, or to calculate absolute target quantities in reference to an appropriate standard curve derived from a series of known dilutions. The Luna Probe One-Step RT-qPCR Kit (No ROX) is identical to the Luna Probe One-Step RT-qPCR Kit but does not contain the universal reference dye and is appropriate for instruments that do not require ROX normalization.
Optimize your RT-qPCR with Luna WarmStart® Reverse Transcriptase
• •
Novel, thermostable reverse transcriptase (RT) improves performance WarmStart RT paired with Hot Start Taq increases reaction specificity and robustness
Experience best-in-class performance
All Luna products have undergone rigorous testing to optimize specificity, sensitivity, accuracy and reproducibility Products perform consistently across a wide variety of sample sources A comprehensive evaluation of commercially-available qPCR and RT-qPCR reagents demonstrates superior performance of Luna products
Make a simpler choice
Convenient master mix formats and user-friendly protocols simplify reaction setup Non-interfering, visible tracking dye helps to eliminate pipetting errors
RT-qPCR targeting human GAPDH was performed using the Luna Universal Probe One-Step RT-qPCR Kit over an 8-log range of input template concentrations (1 μg – 0.1 pg Jurkat total RNA) with 8 replicates at each concentration. Reaction setup and cycling conditions followed recommended protocols, including a 10-minute RT step at 55°C for the thermostable Luna WarmStart Reverse Transcriptase.
NEB’s Luna Universal Probe One-Step RT-qPCR Kit offers exceptional sensitivity, reproducibility and RT-qPCR performance
Products for Sequencing Technologies
The following reagents are being recommended in a number of third party COVID-19 sequencing protocols, including “PCR tiling of COVID-19 virus” in Oxford Nanopore Technologies’ Nanopore Community, and the ARTIC protocol.
Products for Oxford Nanopore Technologies® Sequencing
A number of methods are being developed using the Illumina platform for virus, metagenomic and host sequencing, as well as diagnostics. These include ARTIC-based virus enrichment protocols and novel workflows.
Products for Illumina® Sequencing
• • • • • •
Q5® Hot Start High-Fidelity 2X Master Mix (NEB #M0494) Random Primer Mix (NEB #S1330S) Deoxynucleotide (dNTP) Solution Mix (NEB #N0447) NEBNext® Ultra™ II End Repair / dA-Tailing Module (NEB #E7546) NEBNext Ultra II Ligation Module (NEB #E7595) NEBNext Quick Ligation Module (NEB #E6056)
• • • • • • •
NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645) NEBNext Ultra II RNA First Strand Synthesis Module (NEB #E7771) NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB #E6111) NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) LunaScript RT SuperMix Kit (NEB #E3010)
View Citations for Sequencing and COVID-19
Sequencing and COVID-19 protocols for Nanopore and Illumina sequencing
Featured sequencing protocols
View Protocols
"GMP-grade" is a branding term NEB uses to describe reagents manufactured at NEB’s Rowley facility. The Rowley facility was designed to manufacture reagents under more rigorous infrastructure and process controls to achieve more stringent product specifications and customer requirements. Reagents manufactured at NEB’s Rowley facility are manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards. However, at this time, NEB does not manufacture or sell products known as Active Pharmaceutical Ingredients (APIs), nor does NEB manufacture its products in compliance with all of the Current Good Manufacturing Practice regulations.
GMP-grade reagents for RNA synthesis – Tools to take you from template to transcript
Learn more about the difference between GMP-grade and research-grade reagents
Not sure whether you need research-grade or GMP-grade materials?
For over 45 years, NEB has been a world leader in the discovery and production of reagents for the life science industry. Our enzymology expertise effectively positions us to supply reagents for the synthesis of high-quality RNA – from template generation and transcription, to capping, tailing and cleanup after synthesis. These products are designed and manufactured based on decades of molecular biology experience, so that you can be confident they will work for your application. NEB’s portfolio of research-grade and GMP-grade* reagents enables bench-scale to commercial-scale mRNA manufacturing. Our optimized HiScribe™ kits enable convenient in vitro transcription (IVT) workflows. When it is time to scale up and optimize reaction components, our standalone reagents are readily available in formats matching our GMP-grade offering, enabling a seamless transition to large-scale therapeutic mRNA manufacturing.
We invite you to check out our new COVID-19 Researcher Spotlight Series! Hear from scientists around the world as they discuss their research efforts related to COVID-19, including new protocols and techniques for detection and characterization, as well as how the scientific community is coming together to address this pandemic – all in 19 minutes or less! Learn more at www.neb.com/COVID19.
Additional COVID-19 Resources
Current episodes include:
In our first episode, our NEB podcast host, Lydia Morrison interviews Brian Rabe from Harvard University. Brian’s research usually focuses on retinal development, but the spread of the COVID-19 pandemic motivated him to apply the RT-LAMP technique to improving and advancing coronavirus detection methods.
In our third episode, Lydia interviews NEB Senior Scientist Nathan Tanner, resident expert on isothermal amplification. Learn how Nathan’s group is collaborating with scientists all over the world, working to help develop successful COVID-19 research and testing methods that utilize loop-mediated isothermal amplification.
In our second episode, we interview the Broad Institute’s Feng Zhang, and Omar Abudayyeh and Jonathan Gootenberg from MIT McGovern Institute about the STOPCovid initiative. The STOPCovid test combines isothermal amplification with CRISPR-mediated detection to provide detection of SARS-CoV-2 in about an hour and provides sensitivity comparable to that of RT-qPCR methods.
In our fourth episode, we interview NEB Development Scientist Greg Patton. Greg helped develop the newly released SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit and talks through how this RUO kit works and its intended uses.
Upcoming speakers include:
• Chris Mason, Weill Cornell Medicine • Naama Geva-Zatorsky, Technion –Israel Institute of Technology
Listen to Podcast
View Video
Let us know what you are working on, so we can keep you informed on new COVID-19 related products and publications available from NEB.
Doing COVID-19 related research?
Stay Informed on COVID-19 at NEB
In this webinar, we will provide an overview of several NEB products for DNA amplification, and discuss how they can be used to support COVID-19 diagnostic efforts. We will focus on products that are currently being used by customers, including our Luna RT-qPCR kits and LAMP master mixes. In addition to sharing some of our own efforts to further research in this area, we will be highlighting the use of these products and technologies by discussing selected preprints and other publicly available information. Lastly, we will introduce NEB’s new RUO kit for the rapid detection of SARS-CoV-2 via colorimetric LAMP. Additional details on this product are described above. The simple pink-to-yellow visual readout of viral detection makes it a promising diagnostic approach.
Featured webinar
View Webinar
View archived webinars
Let NEB help you to
For those of you who have not been able to carry out your lab work, we know that the time to return to work will vary by location – some are back in the lab now, or planning to head back in the near future – while others continue to work from home for the time being. Regardless of where you are, we want to help you quickly get back to the important science that you do!
Before returning to the lab
Check and comply with your institutional and local requirements
Identify a co-worker responsible for coordinating and enforcing your new policies
Review and understand any required protocols for health monitoring
Be sure there is adequate stock of PPE available – if not, place an order (avoiding items critical for health care workers)
Once back the lab
|
Pre-plan your day so you can work efficiently while in the lab. Consider performing any non-lab work at home (e.g., writing papers, conference calls, ordering/researching reagent/equipment purchases, etc.)
Stagger worktimes and shifts, including lunch time and breaks
Develop a floorplan that limits the number of people per space, being at least six feet (2 meters) apart, and consider 1-way corridors
Consider break and meeting rooms, as well as lab space
Develop a scheduling system and clean-up strategy for shared equipment, including freezers and refrigerators
Check with delivery rooms for policies on pick-up and drop-off of packages and mail – non-perishable items may need to be quarantined
Develop a plan for visitors, including vendors; discourage in-person visits, unless critical
Develop training materials and protocols for all new procedures; consider making training mandatory
Review and understand travel and commuting policies
Check in with any conferences or tradeshows you were planning to attend
Consider replacing live seminars or trainings with virtual events
Understand the accountability for non-compliance of any safety protocols
Ensure that safety devices, such as fire extinguishers, shower and eye washing stations are checked, clean and working
Flush water lines out, if necessary
Contact your EHS team for disposal of any hazardous materials
Consider a deep clean of the lab periodically
Take stock of reagents and pay attention to expiration dates – reorder, as needed
Move frequently-used equipment to new locations to allow for social distancing
Update software, as needed
Be sure that equipment is calibrated and ready to use – contact manufacturer, if needed
Consider adding additional hand sanitizing areas
Launder lab coats more frequently
Wear protective masks and gloves
Refrain from touching your face
Practice social distancing
Practice proper hygiene – wash your hands frequently
Download printable safety reminders
Request the Reboot Your Bench gift pack
View NEB's selection of fast, high-quality products to help you be more efficient in the lab
Reagents and Equipment
Personal safety
