Loop-mediated isothermal amplification, also known as LAMP, is a simple and cost-effective nucleic acid amplification technique that does not require expensive instruments and helps ensure rapid results and easy outcome interpretation. The technique is cost effective because it is carried out at a constant temperature and does not require a thermal cycler or other expensive equipment. LAMP is used for the detection of various pathogens, including bacteria, viruses, and protozoans that may cause food contamination and plant disease.
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Enzymes and reagents for LAMP and RT-LAMP
Invitrogen LAMP products are available in stand-alone or master mix formats, providing fast, highly sensitive, and specific LAMP reactions with resistance to a variety of inhibitors. They are also compatible with real-time and endpoint detection.
Invitrogen Lyo-ready Bst DNA Polymerase optimizes LAMP reactions
Fast and sensitive detection of various pathogens with engineered Bst DNA polymerase
The Lyo-ready Bst DNA Polymerase is an engineered version of Bst DNA polymerase, large fragment, which shows significantly faster reaction speed, increased sensitivity, and tolerance to inhibitors. Lyo-ready Bst DNA polymerase provides maximum flexibility to optimize your LAMP reaction and work with various types of pathogens, including human adenovirus, measles, SARS-CoV-2, and other pathogens.
Product highlights:
- Fast—amplifies targets in as little as ten minutes
- Sensitive—achieves sensitivity down to 50 copies
- Robust—amplifies even from inhibitor-containing RNA/DNA samples
- Flexible—provides the ability to optimize your LAMP or RT-LAMP reaction
LAMP reaction benefits of lyo-ready Bst DNA polymerase
High speed
The exceptional speed of lyo-ready DNA polymerase provides target detection in as few as ten minutes in a LAMP reaction. Lyo-ready Bst DNA polymerase amplifies DNA and RNA targets faster than other commercially available Bst DNA polymerases in detecting both viral and bacterial pathogens.
Figure 1. Lyo-ready Bst DNA polymerase provides the fastest reaction speed with various DNA targets. Amplification speed was determined in LAMP reactions using adenovirus AdV41 synthetic DNA and Mycoplasma pneumoniae genomic DNA, their respective primer sets and SYTO 9 green fluorescent nucleic acid stain for real-time detection. NEB™ Bst 3.0, NEB™ Bst 2.0 Warmstart, Optigene™ GspSSD 2.0, ArcticZymes™ Isopol Bst+ and Lucigen™ Bst DNA Polymerase were used following the manufacturers’ recommended protocols. Error bars represent standard deviation of reaction speed calculated from three separate experiments for each tested sample.
Lyo-ready Bst DNA polymerase demonstrates fast viral RNA amplification with detection in as few as ten minutes in an RT-LAMP reaction, using SSIV reverse transcriptase for the initial amplification step. When compared to other commercially available Bst DNA polymerases, lyo-ready Bst DNA polymerase shows significantly higher reaction speed.
Figure 2. Lyo-ready Bst DNA polymerase provides the fastest reaction speed with various RNA targets. Amplification speed was determined in RT-LAMP reaction using SARS-CoV-2 synthetic RNA and Measles viral RNA, their respective primer sets, and SYTO 9 green fluorescent nucleic acid stain for real-time detection. NEB™ Bst 3.0, NEB™ Bst 2.0 Warmstart, Optigene™ GspSSD 2.0 and ArcticZymes™ Isopol Bst+ DNA polymerases were used following the manufacturers’ recommended protocols. All reactions have added recommended reverse transcriptase. Error bars represent standard deviation of reaction speed calculated from three separate experiments for each tested sample.
High sensitivity
The high sensitivity of Lyo-ready Bst DNA Polymerase enables amplification from a limited amount of starting material or amplification from samples containing low concentration of the target DNA/RNA. Lyo-ready Bst DNA Polymerase shows significantly higher sensitivity than other commercially available Bst DNA Polymerases. Lyo-ready Bst DNA Polymerase achieves sensitivity down to 50 copies in LAMP reaction.
Figure 3. High sensitivity and reliable amplification from low amounts of DNA. Lyo-ready Bst DNA Polymerase amplifies as low as 50 copies of DNA target in LAMP reaction. Amplification speed was determined using adenovirus AdV41 synthetic DNA and M. pneumoniae genomic DNA, their respective primer sets, and SYTO 9 green fluorescent nucleic acid stain for real- time detection. NEB™ Bst 3.0, NEB™ Bst 2.0 Warmstart, Optigene™ GspSSD 2.0, ArcticZymes™ Isopol Bst+ and Lucigen™ Bst DNA Polymerases were used following the manufacturers’ recommended protocols. Error bars represent standard deviation of reaction speed calculated from three separate experiments for each tested sample.
Figure 4. High sensitivity and reliable amplification from low amounts of RNA. Lyo-ready Bst DNA Polymerase amplifies as low as 50 copies of RNA target in LAMP reaction. Amplification speed was determined using SARS-CoV-2 synthetic RNA and Measles viral RNA, their respective primer sets, and SYTO 9 green fluorescent nucleic acid stain for real- time detection. NEB™ Bst 3.0, NEB™ Bst 2.0 Warmstart, Optigene™ GspSSD 2.0, ArcticZymes™ Isopol Bst+ and Lucigen™ Bst DNA Polymerase were used following the manufacturers’ recommended protocols. Error bars represent standard deviation of reaction speed calculated from three separate experiments for each tested sample.
Tolerance to inhibitors in LAMP assays
RNA and DNA samples may have suboptimal purity even after a thorough sample purification process. Lyo-ready Bst DNA polymerase demonstrates exceptional tolerance to common inhibitors originating from reagents used in RNA/DNA extraction steps or transferred from biological samples.
Table 2. Common LAMP inhibitors and their sources
| Inhibitor | Source |
|---|---|
| Ethanol, salts | Sample prep |
| Heparin, hemin, urea | Blood, urine |
| Humic acid, xylan | Soil, plants |
| Transport media for clinical specimens | UTM |
Figure 5. Lyo-ready Bst polymerase maintains high performance in the presence of inhibitors. Inhibitors were added to LAMP reaction together with 1000 copies of synthetic AdV41 DNA. LAMP reaction speed was analyzed by real-time detection of amplified product using SYTO 9 green fluorescent nucleic acid stain. Relative increase in time to detection shows the change of detection time of the tested sample compared to the control reaction where no inhibitor was added. Error bars represent standard deviation of reaction speed calculated from at least four separate experiments for each tested sample.
Avoid RNA degradation in your LAMP reaction with Lyo-ready RNaseOUT Recombinant Ribonuclease Inhibitor
RNaseOUT Recombinant Ribonuclease Inhibitor is a potent noncompetitive inhibitor of pancreatic-type ribonucleases such as RNase A and is used to avoid RNA degradation in a variety of applications. RNaseOUT Recombinant Ribonuclease Inhibitor is an acidic protein with a molecular weight of ∼52 kDa. RNaseOUT inhibits RNase A, RNase B, and RNase C.
Accelerate pathogen research and surveillance with SuperScript IV RT-LAMP Master Mix
The SuperScript IV RT-LAMP Master Mix is a reverse transcription loop-mediated isothermal amplification (RT-LAMP) based solution for faster and simpler detection of various pathogens, including influenza, measles, S. enterica, S. aureus, SARS-CoV-2, and other pathogens. Our master mix reagents provide maximum flexibility to optimize and accelerate your pathogen research and surveillance.
Product highlights:
- Faster—pathogen detection in as little as five minutes with evolved Bst DNA polymerase
- Efficient—one-step reaction for reverse transcription of RNA to cDNA with SuperScript IV reverse transcriptase
- Sensitive—greater sensitivity and specificity utilizing RNaseOUT recombinant ribonuclease inhibitor and an optimized buffer
- Simple—streamlined workflow: single tube format, only requires a 65℃ heating block
- Flexible—several options for evaluating results, including real-time and endpoint detection methods
RT-LAMP workflow for assay research and development in the study of viral pathogens
Figure 6. Sample RT-LAMP workflow. Example of typical workflow for assay research and development in the study of viral pathogens using purified RNA samples with Invitrogen SuperScript IV RT-LAMP Master Mix.
Lamp reaction benefits of SuperScript IV RT-LAMP Master Mix
High speed
Faster RNA amplification speed for SuperScript IV RT-LAMP Master Mix. Viral pathogen detection in as few as five minutes with evolved Bst DNA polymerase.
Figure 7. SARS-CoV-2 RNA amplification speed. SuperScript IV RT-LAMP Master Mix provides fast RNA target amplification at constant speed even at low target copy number. Amplification speed was determined using synthetic SARS-CoV-2 RNA, the same primer set (from Colorimetric ReadiLAMP™ kit, SARS-CoV-2 Cat. No. A52539), and SYTO 9 green fluorescent nucleic acid stain for real-time detection with Applied Biosystems QuantStudio 5, 6 Flex and 7 Flex Real-Time PCR Systems. NEB™ WarmStart LAMP Kit (DNA & RNA), Optigene™ GspSSD2.0 Isothermal Mastermix, and Lucigen™ LavaLAMP™ RNA Master Mix were used following the manufacturers’ recommended protocols. The values shown are the average of three experiments.
Sensitivity
SuperScript IV RT-LAMP Master Mix demonstrated robust detection at low RNA copy numbers, detecting as low as 30 copies in less than ten minutes.
Figure 8. Detecting low SARS-CoV-2 RNA copy numbers. SuperScript IV RT-LAMP Master Mix amplifies as low as 30 copies of RNA target in under 10 minutes. Amplification speed was determined using synthetic SARS-CoV-2 RNA, same primer set (from Colorimetric ReadiLAMP™ kit, SARS-CoV-2 Cat. No. A52539) and SYTO 9 green fluorescent nucleic acid stain for real-time detection with Applied Biosystems QuantStudio 5, 6 Flex and 7 Flex Real-Time PCR Systems. NEB™ WarmStart® LAMP Kit (DNA & RNA), Optigene™ GspSSD2.0 Isothermal Mastermix, and Lucigen™ LavaLAMP™ RNA Master Mix were used following the manufacturers’ recommended protocols. The values shown are the average of three experiments.
Flexibility
SuperScript IV RT-LAMP Master Mix provides several options for evaluating results, including real-time and endpoint methods.
Figure 9. Detection of the viral RNA target by the real-time fluorescent RT-LAMP assay. RT-LAMP was performed using a real-time PCR instrument and SYTO 9 stain. The reaction time was 60 minutes. Two blue curves demonstrate an early amplification signal in positive reactions (<15 min). The dark blue curve represents amplification of the purified clinical RNA, and the light blue curve corresponds to the synthetic viral RNA amplification. The grey curve indicates late amplification signal in the no template control.
DNA/RNA applications
SuperScript IV RT-LAMP Master Mix also detects DNA targets, enabling a wide range of applications for your research and surveillance of pathogens.
Figure 10. DNA target amplification. SuperScript IV RT-LAMP Master Mix can be used to amplify DNA targets while demonstrating exceptional amplification speeds. Amplification speed was determined using DNA plasmid with SARS-CoV-2 fragment, same primer set (from Colorimetric ReadiLAMP™ kit, SARS-CoV-2 Cat. No. A52539) and SYTO 9 green fluorescent nucleic acid stain for real-time detection with Applied Biosystems™ QuantStudio 5, 6 Flex and 7 Flex Real-Time PCR Systems. NEB™ WarmStart® LAMP Kit (DNA & RNA), Optigene™ GspSSD2.0 Isothermal Mastermix, and Lucigen™ LavaLAMP™ RNA Master Mix were used following the manufacturers’ recommended protocols. The values shown are the average of three experiments.
Developing a LAMP assay?
Invitrogen stand-alone enzymes include the polymerase, buffer, and other additional components to ensure maximal flexibility in reaction setup and the feasibility of developing assays for LAMP and RT-LAMP applications. They can be customized in the glycerol-free lyo-ready format.
Learn more about our lyophilization products at www.thermofisher.com/lyo-ready or request a quote for custom commercial supply.
Ordering information for LAMP assay enzymes and reagents
Application notes
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