The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is designed to provide a simple, one-step solution for LAMP/RT-LAMP of DNA or RNA targets. LAMP and RT-LAMP are commonly used isothermal amplification techniques that provide rapid detection of a target nucleic acid using LAMP-specific primers (supplied by the user) and a strand-displacing DNA polymerase. This master mix features a blend of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized LAMP buffer solution. Both Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase have been engineered for improved performance in LAMP and RT-LAMP reactions. The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is fully buffered and compatible with different sample types, enabling multiple detection methods including turbidity detection, real-time fluorescence detection (when used with LAMP fluorescent dye) and end-point visualization such as colorimetric detection via a metal indicator (e.g., hydroxynaphthol blue.)
Figure 1: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with multiple detection methods
Amplification of target RNA or DNA by the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) can be confirmed using a variety of real-time (A) and endpoint (B) detection methods. Some of these detection modalities include fluorescence, non-pH-based colorimetric (e.g., hydroxynaphthol blue), turbidity or agarose gel electrophoresis. For real-time fluorescence detection, the master mix is available as a kit (NEB #E1708) that includes 50X LAMP Fluorescent Dye, which can be monitored in the SYBR®/FAM channel of most real-time PCR instruments.
The inclusion of dUTP and thermolabile UDG in the mix reduces the possibility of carryover contamination, where unintended product of a previous amplification can serve as the substrate of a subsequent reaction. Thermolabile UDG is completely inactivated at temperatures above 50°C, thereby having no effect on the reaction.
Figure 2: LAMP/RT-LAMP master mixes and sample type considerations
NEB's pH-based colorimetric LAMP master mixes with UDG (NEB #M1804) or without UDG (NEB# M1800) are weakly buffered to allow for visual detection of amplification using a pH-sensitive dye. However, the low buffering capacity required to generate the pink to yellow color change limits sample compatibility with the pH-based colorimetric mixes, as highly buffered sample inputs or acidic samples may impact the color change. The multi-purpose LAMP/RT-LAMP 2X Master Mix with UDG (NEB #M1708, NEB #E1708) or without UDG (NEB #E1700) is fully buffered and can more readily tolerate these types of sample inputs, making it compatible with various detection modes including fluorescence or other colorimetric dyes (e.g., hydroxynaphthol blue).
Figure 3: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) provides robust detection of human DNA and RNA targets
RT-LAMP (RNA targets) or LAMP (DNA targets) experiments were performed with NEB #M1700: WarmStart LAMP 2X Master Mix, which is the master mix in the NEB #E1700 kit that does not contain dUTP/UDG, and NEB #M1708: WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG). Reactions containing 1X LAMP primers and 1X LAMP Fluorescent dye were set up in quadruplicate over three logs of total Jurkat RNA or Jurkat DNA (10 ng to 0.1 ng) in 96-well, 25 µl reactions. Control reactions without template (NTC) were also evaluated. Reactions were incubated at 65°C for 40 minutes and fluorescence was monitored every 15 seconds in the SYBR/FAM channel of a real-time thermocycler (Bio-Rad® CFX96). Each dot represents the time at which the fluorescence signal for a single reaction crosses the instrument-defined threshold. All four replicates were detected at each template input unless otherwise indicated (note that dots frequently overlap given similar detection time for the replicates). Overall, similar performance was observed for both NEB #M1700 and NEB #M1708 at each template input. No amplification was observed in any of the no template control reactions.
Figure 4: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with automated reaction assembly
LAMP assays targeting synthetic SARS-CoV-2 RNA were assembled either manually or with the TEMPEST® liquid handling platform (96-well, 25 µl reactions). The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction) or no template (NTCs) as indicated. Reactions were incubated at 65°C for 40 minutes and monitored with 1X LAMP Fluorescent dye in the SYBR/FAM channel of a real-time instrument (Bio-Rad CFX96). Similar time to detection and LOD results were observed for both reaction assembly methods.
Figure 5: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with non-pH-based colorimetric detection (e.g., hydroxynaphthol blue)
The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) was used to amplify synthetic SARS-CoV-2 RNA using a dual primer mix that targets the N and E genes in the presence of 0.12 mM of hydroxynaphthol blue, a colorimetric metal indicator. The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction), negative samples (human total RNA alone) or no template (NTCs), as indicated. The 25 µl reactions were incubated at 65°C for 45 minutes and then visually inspected.
A. All positive samples gave a positive result with robust color change from purple to blue, including detection in all samples at a low input of 50 copies per reaction (n = 20).
B. No color change was observed for numerous replicates of negative samples (n = 32).
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