09/03/2026

Fast, Simple, Flexible: Introducing our
New RPA basic Kit – The Perfect Basis
for Customized RPA Lateral Flow Assays

Rapid molecular testing does not always require complex instrumentation. With our new RPA kit, Milenia Biotec provides researchers and assay developers with a simple and flexible solution for fast isothermal DNA amplification, offering an ideal basis for optional integration into an RPA lateral flow assay.

Why Recombinase Polymerase Amplification (RPA)?

RPA enables rapid DNA amplification at a constant, relatively low temperature. Unlike conventional PCR, it does not require repeated thermal cycling, reducing the need for complex laboratory equipment and simplifying the overall workflow. These characteristics make RPA particularly attractive for applications where fast results, flexibility, and ease of use are important.

Developing an RPA-based assay often means balancing reaction speed, robustness and ease of use. Researchers need reagents that integrate smoothly into their workflow and allow them to focus on assay development rather than reaction setup.

This is exactly where the new Milenia RPA basic Kit comes in.

Introducing the Milenia RPA basic Kit

The Milenia RPA basic Kit is designed to make isothermal DNA amplification straightforward, fast, and easy to integrate into existing assay development workflows. By combining the key advantages of RPA with a user-friendly kit format, it provides researchers and assay developers with a flexible basis for rapid nucleic acid amplification.

Key benefits include:

Fast amplification – DNA amplification within a short reaction time
Low-temperature operation – reactions run at a constant temperature of approximately 37–42 °C
Freeze-dried reaction mix—can be stored at room temperature
Simple workflow – ready-to-use mix, no thermal cycling or complex instrumentation required
Flexible assay development – compatible with target-specific primer design and different downstream detection approaches
Suitable for lateral flow readout – appropriately labeled amplicons can be integrated into rapid visual detection workflows

The workflow is designed to keep amplification straightforward. After adding the target-specific primers and sample to the reaction mix, amplification takes place at a constant temperature. The amplified product can subsequently be detected using an evaluation platform of choice (agarose gel electrophorese, lateral flow (NALFIA), etc).

Workflow Graphic RPA Lateral Flow Assay (NALFIA)

Figure 1: Workflow of RPA assay evaluated by lateral flow

RPA + Lateral Flow Assay (LFA)

Did you know that the success story of Recombinase Polymerase Amplification is closely linked to our HybriDetect test strips? Back in 2006, the very first pioneering RPA study by Piepenburg O. et al. combined this technology with our Milenia HybriDetect lateral flow detection system. This combination of RPA and lateral flow enables a rapid and easy-to-interpret visual readout without the need for sophisticated analytical instruments showing a real point-of-need solution.

Converting an existing RPA assay to lateral flow evaluation is remarkably simple. No changes to the primer sequences are necessary. This is made possible by using specially labelled primers, which later interact with the test strip. More detailed information on how this works can be found in the article Recombinase Polymerase Amplification & Lateral Flow.

Practical example: Detection of Levilactobacillus brevis

We were able to demonstrate the compatibility of the RPA basic Kit with our HybriDetect test strips using the detection of Levilactobacillus brevis as an example. L. brevis plays the major role in the spoilage of beverages and is therefore of economic interest to the beverage industry.

The Milenia RPA basic Kit was used to detect Levilactobacillus brevis. Primers were designed using free primer design tools such as Primer3. To make the amplificates visible on the strip, the forward and reverse primers were labelled with biotin and FAM at their 5′ ends, respectively.

Primer	Sequence (5‘ → 3‘)	5´-Label
L. brevis Forward Primer	32 bp primer	Biotin
L. brevis Reverse Primer	30 bp primer	FAM

The RPA master mix consists of one LyoBead RPA basic, 25 µl of ready-to-use 2X RPA Reconstitution Buffer X, 15.7 µl of nuclease free water, 2.4 µl of primer mix (2.5 µM primer each), 2 µl of sample and 2.5 µl of 20X RPA Reaction Initiator. The RPA mixture was incubated for 20 minutes at constant temperature. The reaction was then stopped by heating to 95 °C for 10 minutes (optional).

The reaction was evaluated using agarose gel electrophoresis (AGE) and lateral flow test strips, respectively. A 1.5 % gel was used for the AGE where 5 µl of the RPA reaction were loaded. The evaluation with the HybriDetect – Universal Lateral Flow Test Strips was performed by applying 1 µl of the RPA reaction and 80 µl of running buffer to the test strip. The evaluation was performed after 5 minutes. If L. brevis is present in a sample, a part of the DNA is amplified which becomes visible through corresponding bands in the gel or lines on the test strip (see figure).

Figure 2: Evaluation of the RPA reaction (42 °C, 20 minutes) for the detection of Levilactobacillus brevis using agarose gel electrophoresis (1.5% gel) and LFA (Milenia GenLine HybriDetect test strip, REF: MGHD 1). NC = negative control; PC = positive control; M = 100 bp Marker

RPA is a highly process-driven method for amplifying DNA. This results in a very fast amplification of the target DNA but can quickly lead to false-positive signals too. This is particularly noticeable when using lateral flow test strips for analysis. Adjusting the primer concentration is one way to counteract this (see figure). In this case higher concentrations (> 0.24 µM) showed false positive signals in negative controls. Lower concentration (> 0.048 µM) couldn’t generate a positive signal in the positive controls. A good working concentration for our example assay was 0.12 µM. Here we were able to get a positive signal while the negative control stayed negative.

Figure 3: Investigation of various primer concentrations (0.48 µM – 0.048 µM) for RPA reaction for differentiation between positive and negative samples. NC = negative control; PC = positive control

To determine the best reaction temperature, we firstly tested a temperature range between 30 and 50 °C at low template concentration. The results indicate an optimal incubation temperature of 42 °C for our example assay. In general, the optimal temperature can vary in a small range for each specific assay, test setup or heating conditions. Overall, our RPA basic Kit has its optimal conditions between 37 and 42 °C.

Figure 4: Signal intensity of the RPA reaction (20 minutes) for the detection of Levilactobacillus brevis at different reaction temperatures (30-50 °C) using LFA (Milenia GenLine HybriDetect test strip, REF: MGHD 1). NC = negative control. n=1

To give you an idea of how our kit performs, we compared it to the current standard among RPA kits under identical conditions (primer concentration, sample material, incubation times). The only difference was the reaction temperature, which is 42 °C for our kit and 39 °C for the competitors kit (according to their kit’s specifications). Sample material for this comparison was a purified PCR DNA amplicon covering the target sequence. As you can see below there is no difference between our and the competitor’s kit. Both kits amplify the target DNA and can detect the 10^-3 dilution equally well.

Figure 5: Sensitivity of the Milenia RPA basic Kit (left) compared to a competitors RPA basic Kit (right). Sample material was a purified PCR DNA amplicon dilution series which covered the target sequence. The evaluation was done using LFA (Milenia GenLine HybriDetect test strip, REF: MGHD 1). PC = positive control; NC = negative control. The white arrow marks the LOD.

Compared with agarose gel electrophoresis, lateral flow detection enables rapid, equipment-light and easy-to-interpret visual readout, making it particularly attractive for decentralized and point-of-need molecular testing.

Using our HybriDetect strips also gives you another advantage as you can see below. In this case we compared both evaluation techniques while we applied 5 µl of RPA mixture to the 1.5 % gel and 1 µl to the lateral flow strip. As you can see our test strips are at least 10 times more sensitive than evaluation via agarose gel electrophoresis.

Figure 6: Direct comparison of the two evaluation techniques agarose gel electrophoresis and our lateral flow test strips (Milenia GenLine HybriDetect test strip, REF: MGHD 1). 5 µl RPA product were applied to the 1.5 % agarose gel while 1 µl was applied to the test strip. The white arrow marks the LOD.

Summary

Within a short period of time, RPA-LFA detection was established using the Milenia RPA basic Kit and the HybriDetect Universal test strip. The total detection time from sample addition to result output is 25 minutes. Only a (mobile) heating block is required to perform the detection, which enables use at the point of need. By combining RPA and lateral flow detection from a single source, we provide you with the tools to develop a customized molecular detection assay tailored to your specific application.