The LavaLAMP™ DNA Component Kit is designed for loop-mediated isothermal amplification (LAMP) assay development. This kit contains a unique, highly processive DNA polymerase enzyme with strong strand displacement activity, making it well suited to LAMP and other isothermal amplification assays.
The enzyme in this kit is identical to the enzyme present in the LavaLAMP™ DNA Master Mix, but the individual reagent format of this kit, as opposed to the master mix format, gives you complete control of each LAMP assay and full optimization capabilities. Importantly, this kit is an exact deconstruction of the LavaLAMP® DNA Master Mix, and when reactions are setup using the initial Experimental reaction conditions outlined in the Component Kit User Manual, the composition of that reaction formulation matches the composition of the Master Mix-based reaction.
To aid in assay development and future lyophilization of LavaLAMP®-based reactions, all components in this kit are compatible with lyophilization. This feature means that reactions will not have to be reformulated or reoptimized to remove components that hinder lyophilization such as glycerol and betaine.
For more information on loop-mediated isothermal amplification, please look at the following recorded webinar, “Loop-Mediated Isothermal Amplification (LAMP): Assay Development Challenges and Solutions.”
Figure 1. Performance comparison of the LavaLAMP™ DNA Component Kit and the LavaLAMP™ DNA Master Mix. LAMP reactions (8 replicates per test) were set up using the indicated kits and targets with the same concentrations of Target-Specific Primer Mixes (1X) and the same target input amounts. The LavaLAMP DNA Component Kit reactions were formulated to 1X LavaLAMP DNA Buffer, 10 mM MgSO4 and 0.8mM dNTPs (each) to match the final formulations of the LavaLAMP DNA Master Mix reactions. Green Fluorescent Dye was included in all reactions. Reactions were run on a CFX96 Thermal Cycler (Bio-Rad) at the following temperatures - M13mp18, 74°C and tcdA, 69°C and fluorescence was measured during the course of the 60-minute reactions to determine TTR. NTC = No Target Control.
Figure 2. Performance comparison of the LavaLAMP™ DNA Component Kit vs. Competitor Kits. LAMP reactions were set up using the indicated kits according to manufacturer’s recommendations. Target DNA (C. difficile) at varying input amounts, tcdA target LAMP primers, and Green Fluorescent Dye (LavaLAMP Kit) were included in all reactions. Reactions were run on a CFX96 Thermal Cycler (Bio-Rad) at the following temperatures: LavaLAMP; 69°C; other kits at the recommended 65°C and fluorescence was measured over 60 minutes to determine the TTR. NTC = No Target Control.
Figure 3. Analysis of operator to operator consistency. On the same day, six replicate LAMP reactions were set up per condition per by three different operators using the indicated amounts of target DNA M13mp18, M13mp18 target LAMP primers, and Green Fluorescent Dye. Reactions were run on a CFX96 Thermal Cycler (Bio-Rad) at 74°C and amplification was monitored in real-time. Results were analyzed to determine the TTR and averaged. NTC = No Target Control.
Figure 4. Analysis of day to day assay consistency. On three different days, six replicate LAMP reactions were set up per condition using the indicated amounts of target DNA M13mp18, M13mp18 target LAMP primers, and Green Fluorescent Dye. On each day the reactions were run on a CFX96 Thermal Cycler (Bio-Rad), amplification was monitored in real-time, and then results were analyzed to determine the TTR. Replicates were averaged and presented. NTC = No Target Control.
Figure 5. Assessing the importance of primer design and reaction temperature on LAMP assay performance. Three primer sets targeting M13mp18 were designed using the LAMP Designer software (Premier Soft) and screened across a temperature range of 68°C – 74°C using the LavaLAMP™ DNA Component Kit at the recommended 1X, default reaction conditions. Duplicate assays were run for each test, and real-time detection was performed on a CFX96 Thermal Cycler (Bio-Rad) using Green Fluorescent Dye. NTC = No Target Control. Primer Set 3 provided the fastest positive Time to Result with the least amount of background (longest TTR for NTC reactions). Within Primer Set 3, 74°C provided the best resolution between the positive and negative samples.
Note: Exact temperatures were set by the CFX96 Thermal Cycler instrument software when the range was selected. Specific temperatures tested will depend on the real-time instrument used.
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