Loop-Mediated Isothermal Amplification (LAMP)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.