The LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) is offered in a lyophilized format, allowing for ambient/room temperature shipping and storage before use. It is a simple, one-step solution for real-time fluorescent detection of LAMP/RT-LAMP reactions. LAMP and RT-LAMP are commonly used isothermal amplification techniques that provide rapid detection of a target nucleic acid using LAMP-specific primers and a strand-displacing DNA polymerase. Featuring a blend of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized buffer solution, this mix offers improved performance. LAMP Fluorescent Dye at 0.5X concentration is included to enable detection of amplification via real time fluorescence. The inclusion of dUTP and thermolabile UDG in the master 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.
This product is available in 2 x 50 reaction glass vials or a 96-well plate (1 reaction per well). The plate features twelve partially-attached 8-well strip tubes, allowing for use of the entire plate or partial consumption. The yellow shell frame adaptor supports the use of the plate in a variety of common real-time PCR instruments. Both formats can be customized. Additionally, should alternative detection methods be desired (CRISPR-Cas, end-point visualization such as colorimetric detection via a metal indicator (e.g., hydroxynaphthol blue.), etc.), please contact us for more information.
Figure 1: LyoPrime LAMP offers a robust, versatile LAMP/RT-LAMP option for fluorescence detection in a shelf-stable lyophilized format
The LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) (NEB #L4401) offers the same versatile features as the WarmStart® Fluorescent LAMP/RT-LAMP Kit (with UDG) (NEB #E1708) in a lyophilized format that can be shipped and stored at room temperature. It is available in glass vials or a 96-well plate.
Figure 2: The LyoPrime WarmStart Fluorescent LAMP/RT-LAMP 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 #E1708: WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG) using 0.5X LAMP Fluorescent Dye, and NEB #L4401: LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG). Reactions containing 1X LAMP primers 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 #E1708 and NEB #L4401 at each template input. No amplification was observed in any of the no template control reactions.
Figure 3: LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) enables strong detection of various infectious disease targets
RT-LAMP reactions (25 μl) were performed at 65°C for 30 minutes using 1X primers targeting the indicated viral RNA gene at 5000, 500, or 50 copies per reaction or no template (NTC) as indicated. Fluorescence was monitored every 15 seconds in the SYBR/FAM channel of a real-time thermocycler (Bio-Rad® CFX96). A) Performance was evaluated in quadruplicate for 5000 and 500 copies or twelve replicates for 50 copies of ATCC® synthetic viral RNA (Chikungunya Virus: VR-3246SD; Dengue-2 Virus: VR-3229SD; Zika Virus: VR-3252SD). B) Performance was evaluated in quadruplicate for 5000 and 500 copies or thirty-six replicates for 50 copies of synthetic SARS-CoV-2 RNA. A summary of results from analogous testing conducted by 3 users on SARS-CoV-2 RNA is shown in the table. Each dot represents the time at which the fluorescence signal for a single reaction crosses the instrument-defined threshold. All replicates were detected at each template input for these evaluations unless otherwise indicated (note that dots frequently overlap given similar detection time for the replicates). No amplification was observed in any of the no template control reactions. The LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) exhibited robust detection for each infectious disease target.
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