What Is RT-LAMP and How Does the Kit Work?

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that uses a strand-displacing DNA polymerase and a set of four to six specially designed primers to amplify a target sequence with high specificity under isothermal conditions, eliminating the need for a thermal cycler. When coupled with reverse transcription (RT), the technique can directly amplify RNA targets, including viral genomes and messenger RNAs.

The RT-LAMP Isothermal Amplification Kit (with Fluorescent Dye, Lyophilized) integrates a reverse transcriptase and a strand-displacing polymerase into a single lyophilized reaction pellet. Upon rehydration with the sample and buffer, the enzyme mix converts the RNA template into cDNA and immediately initiates LAMP amplification. A fluorescent intercalating or sequence-specific dye included in the formulation produces an increasing fluorescence signal as amplification products accumulate, enabling real-time monitoring with a compatible fluorescence reader or simple visual inspection under UV light.

The lyophilized format stabilises all enzymatic and chemical components at room temperature, reducing cold-chain dependence during shipping and storage.

Recommended Protocol and Reaction Setup

Rehydration: Briefly centrifuge the lyophilized tube before opening. Add the recommended volume of nuclease-free water or provided rehydration buffer directly to the pellet and mix gently by pipetting. Avoid vortexing to prevent enzyme denaturation.

Primer and Sample Addition: Add your pre-designed RT-LAMP primer set (typically comprising FIP, BIP, F3, B3, and optionally LF and LB primers) and RNA template to the rehydrated master mix. Primers are not included in the kit and must be designed or sourced separately to match your target sequence.

Reaction Conditions: Incubate the reaction at a constant temperature, typically in the range of 60–65 °C, for 30–60 minutes. The optimal temperature and time should be validated empirically for each primer set and target.

Detection: Monitor fluorescence in real time using a compatible isothermal fluorescence reader, or read the endpoint fluorescence after incubation. A positive result is indicated by a significant increase in fluorescence relative to a no-template negative control.

Controls: Always include a no-template control (NTC) and, where possible, a positive RNA control to validate each run.

Storage, Stability, and Handling

Lyophilized Kit Storage: Store the lyophilized reagent according to the conditions specified on the product label and Certificate of Analysis. The lyophilized format is designed for improved thermal stability; however, the recommended storage temperature should be followed to ensure optimal performance over the stated shelf life. Consult the product documentation for specific temperature requirements.

After Rehydration: Once rehydrated, the master mix should be used promptly. Avoid repeated freeze-thaw cycles of the rehydrated material, as this can reduce enzyme activity. If the rehydrated mix cannot be used immediately, store on ice and use within the timeframe specified in the protocol.

RNA Sample Handling: RNA is highly susceptible to degradation by RNases. All work with RNA should be performed using RNase-free consumables, water, and surfaces. Wear gloves at all times and change them frequently to prevent RNase contamination from skin.

Contamination Prevention: LAMP amplification produces very large amounts of amplicon. To prevent carryover contamination, maintain strict physical separation between pre- and post-amplification areas, and use aerosol-barrier pipette tips throughout.

Troubleshooting Common Issues

No Signal in Positive Control: Verify that the reaction temperature is accurate and stable. Check that the correct volume of rehydration buffer was used and that the pellet was fully dissolved. Confirm that primers were added at appropriate concentrations and that the RNA positive control is intact and undegraded.

Signal in No-Template Control (False Positive): LAMP is prone to non-specific amplification and aerosol contamination due to its high sensitivity. Ensure strict separation of pre- and post-amplification areas. Use fresh, RNase-free water for rehydration. Redesign primers if non-specific amplification persists.

Weak or Delayed Signal: Optimise primer concentrations (typically FIP and BIP at higher concentrations than outer primers). Ensure RNA input is within the recommended range and of sufficient quality. Suboptimal incubation temperature or inhibitors in the RNA extract may reduce efficiency.

Inconsistent Results Between Replicates: Ensure thorough mixing after rehydration and primer addition. Pipette carefully to avoid introducing air bubbles. Use a calibrated heat block or water bath with uniform temperature distribution.

Ordering, Compliance, and Support

How to Order: The RT-LAMP Isothermal Amplification Kit (with Fluorescent Dye, Lyophilized) is available from Aladdin Scientific under SKU R1547530. Orders can be placed through the Aladdin Scientific website, by contacting your local sales representative, or via institutional procurement channels. Please confirm pack size and quantity requirements at the time of ordering.

Regulatory Status: This product is intended for research use only (RUO) and is not approved, cleared, or licensed for diagnostic, therapeutic, or clinical use. Users are responsible for validating the product for their specific application in accordance with applicable regulations.

Technical Support: For application-specific guidance, protocol optimisation advice, or troubleshooting assistance, please contact Aladdin Scientific technical support. Provide the product SKU, lot number, a description of your experimental setup, and the issue observed to facilitate a prompt response.

Safety Data Sheet (SDS): An SDS for this product is available on the Aladdin Scientific website and can be requested from customer service. Review the SDS before use and follow all applicable laboratory safety regulations when handling reagents.