Team:Leiden/Proof Of Concept

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Rapidemic

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Proof of Concept

Our experimental results provided preliminary proof that the new detection technology can detect and report the presence of target DNA. Here we show how the technology holds the potential to be further optimized into a rapidly adaptable point-of-care diagnostic tool. We designed and constructed a prototype that forms a bridge between the molecular basis of the technology and the hardware design of a test kit.

Introduction

Our technology integrates three bio-molecular reactions and applies them as a novel method to detect the presence of a target sequence. The three reactions were coupled in a serial manner by using the product of each preceding reaction as a component for each subsequent reaction. After a few rounds of optimization, we performed a proof of concept experiment to show how the three reactions can successfully be connected to detect, amplify, and report the presence of a targeted DNA sequence. The experiment provided the preliminary proof that our sequence detection scheme can work as intended. Subsequently, we designed and constructed a prototype test kit to show how the technology holds the potential to be further optimized into a rapidly adaptable point-of-care diagnostic tool.

Our detection mechanism consist of the following three reactions:
1) Recombinase polymerase amplification (RPA): isothermal exponential amplification of a target sequence using target-specific primers.
2) Linear strand-displacement amplification (LSDA): production of a single-stranded DNA sequence, a DNAzyme, that functions as a reporter in reaction three.
3) DNAzyme-catalyzed oxidation reaction that yields a color change.

Read more about our detection mechanism on the Engineering page.

Design

The design of the prototype consisted of 3x3 reactions to enable subsequent amplification and detection of (1) a test sample, (2) a negative control and (3) a positive control (Fig. 1). The three reactions that are performed for every sample are an RPA reaction, an LSDA reaction and a DNAzyme-catalyzed color reaction. These reactions take place in small wells with a volume of 20 µL for RPA, 10 µL for LSDA and 100 µL for the color reaction. The reaction wells are enclosed in a light-impermeable box to prevent contamination of the reagents and to protect the light-sensitive substrate of the color reaction (TMB)1 from degradation. The prototype can be used by adding the test sample to the well of the first reaction (RPA), followed by two cycles of incubation and transfer to the next reaction, until the last reaction leads to the colorimetric test result. Through the window on the top, the color change can be observed by the naked eye.

Fig. 1 Design of the prototype. The size of the wells is not to scale.

    The reaction wells

  • The RPA wells contain all RPA reagents* except for the target DNA and magnesium acetate (MgOAc). The addition of these components to the wells initiate the RPA reaction.
  • The LSDA wells contain all LSDA reagents except the template DNA. The reaction will start upon adding a fraction of the RPA product, which contains the LSDA template.
  • The color reaction wells contain all oxidation reagents except the catalyst (DNAzyme) and the start solution (TMB and H2O2). The product of the LSDA reaction, which contains the DNAzyme, and the start solution are added to initiate the reaction.

    • *Protocols are on the Experiments page.

    The controls

  • The positive control differs from the test sample in that it contains a synthetic target sequence in place of a (human) sample. In the final kit, the signal in the positive control sample will indicate that the enzymes have sufficient activity after manufacturing, transport and storage. For instance, the signal from the positive control indicates that any suboptimal transport and storage conditions of the kit did not hamper with the enzymes' performance.
  • The negative control lacks the target DNA sequence. This control confirms that there is a visible difference in color between a positive and a negative sample. It was included because we observed that the color of a negative reaction changed to very light blue due to the low oxidizing activity of hemin. We aim to optimize the reaction conditions such that the negative sample remains transparent and this negative control can be removed in the final test kit.

Construction

After making the design, we constructed the prototype. The prototype was made from a 96-well plate, enclosed in a custom-made light-impermeable box (Fig. 2a). The prototype has a slider that can be opened to create a small window. Through the window, we can observe three wells, each containing the reagents for the color reaction. From left to right, the wells represent the test sample, the negative control and the positive control.

Here the RPA and the LSDA reactions were performed in PCR tubes and the color reaction was performed in the prototype device. The color reaction was initiated as soon as the LSDA product and the start solution, containing the substrates of the color reaction, were added through the window into the wells. The box was closed immediately to prevent light-induced bias to the color reaction. Every couple of minutes, the slider was opened shortly to make a snapshot of the colors. After 10 minutes, a clear blue color was visible in the wells of the sample and the positive control (Fig. 2b). The transparent color of the negative control clearly showed the difference in color between the positive and the negative reaction. All in all, the prototype showed that the result of the three subsequent reactions can be obtained within 10 minutes without the need for special equipment.

The total reaction time was 70 minutes, consisting of 20 minutes for the first reaction (RPA), 40 minutes for the second reaction (LSDA), and 10 minutes for the color reaction. However, follow-up experiments have shown that the incubation period for RPA can be reduced to 8 minutes at 30 ºC (Results section Fig. 4). Alternatively, our empirical model that simulates the underlying reaction kinetics predicted that the total reaction time can be reduced to 46.5 minutes, consisting of 15 minutes of RPA reaction, 10 minutes of LSDA reaction, and 21.5 minutes of color reaction.

Fig. 2 Rapidemic prototype. a) First Rapidemic prototype made from black cardboard. b) Snapshots of three oxidation reactions with LSDA product (sample), phosphate citrate buffer (- control) and synthetic DNAzyme (+ control). Stop solution was added after 28 minutes, changing the blue color to yellow. After 1 hour and 21 minutes, the negative control had turned yellow as well, indicating the TMB in the negative control was oxidized as well and thus the time window of readout was exceeded.

Panel 1. Changes compared to the initial design

  1. Because we could not use (human) samples due to safety reasons, the positive control in the prototype would have been similar to the sample reaction; they would both contain a synthetic DNA template. Therefore, the positive control here contained a synthetic DNAzyme (EAD2+3'A) in place of the LSDA product.
  2. For the negative control, we did not perform an RPA reaction that lacks the template DNA, because such reactions showed high false-positive signals in previous results (Results section Fig. 10). Instead, the negative control in the prototype contained buffer in place of RPA-LSDA product. Further research should focus on reducing the false-positive signals to allow the negative control as designed initially.
  3. The RPA and the LSDA reactions were performed in PCR tubes and only the color reaction was performed in the prototype device. The reason for this was that the reaction volumes for RPA (20 µL) and LSDA (10 µL) were not suited for the 96-well plate. A future prototype would contain smaller wells for these two reactions.

Cost estimation

The reagent costs were $ 1.45 per reaction, consisting of $ 1.30 for the RPA reaction, 15 cents for the LSDA reaction and 0.05 cents for the color reaction (Table 1). The greatest contributor to the total cost is the commercial TwistAmp Basic RPA kit (TwistDx) with 89% (Fig. 3). Other expensive reaction components are the dNTPs and the nickase (both 6 cents). The costs for the nickase were already reduced 40-fold by decreasing the concentration of nickase and the LSDA reaction volume (Results section Fig. S4-5). Future work may focus on integrating the first two reactions (RPA and LSDA) into a one-pot reaction, which would reduce the amount of reagents needed in the resulting detection kit (i.e. dNTP, polymerase) and may also a shorten the reaction time.

Click here to download table 1.

The cost of our technique is similar to low-cost commercial amplification kits (Fig. 4). The costs of the commercial kits were normalized to a reaction volume of 20 µL since this allows comparison with our technology which has an RPA reaction volume of 20 µL.

Fig. 3 Cost fraction per component in our detection technology.

Fig. 4 Costs per reaction of 20 µL of commercial RT-PCR, LAMP and RPA assays compared to our technology. Costs retrieved on October 3rd 2020 from references 4-12.

Test different material (paper)

The color change of the prototype described above was detected in a liquid solution. However, detection in solution is not the only possibility; since the RPA reaction can be performed on paper2, 3, it might be interesting to test if our detection method can also be performed on paper. Here, we confirmed that the color reaction was clearly observable by the naked eye when performed on blotting paper (Fig. 3). Visit the Hardware page to read more about different types of materials that can be used in RPA-based detection kits.

Fig. 3 GQ-catalyzed TMB oxidation reaction on blotting paper. a) TMB oxidation reaction performed with 10 µL DNAzyme (EAD2+3'A, 1 µM). Instead of pipetting the reagents in the wells of a 96-well plate, the reagents were pipetted on blotting paper. b) After the addition of sulfuric acid as stop solution, the blue color changed into a yellow color.

Conclusion

Here we demonstrated how the three chemical reactions of our detection technique can be implemented into a prototype device to show the potential of the technique to be further optimized into a rapidly adaptable point-of-care diagnostic tool. In contrast to the first and the last reaction, which were fast and operated at low temperatures (8 min at 30 ºC and 10 min at room temperature for the first and last reaction, respectively), the second reaction was rather slow and was performed at a higher temperature (40 min at 55 ºC). This resulted in a total reaction time of 70 minutes. Future optimization of this reaction in concert with the other two may shorten the overall reaction time; our empirical model that simulates the underlying reaction kinetics of our detection method predicts that the total reaction time can be reduced to 46.5 minutes, consisting of 15 minutes of RPA reaction, 10 minutes of LSDA reaction, and finally 21.5 minutes of GQ-mediated TMB oxidation reaction. The cost of the technology was $ 1.45 per sample, which similar to low-cost commercial amplification kits. Important future work is the integration of the first two reactions into a one-pot reaction. This will not only reduce the reagent costs and the reaction time, but it would also greatly simplify the hardware design, as fewer reaction chambers and transfer steps would be needed. Further investigation into automation of the flow of sample through the chain of reactions and the type of materials could turn this prototype into an easy-to-use device. For more insights on how this can be realized, please visit the Hardware page.

References

  1. National Center for Biotechnology Information. PubChem Compound Summary for CID 41206, 3,3',5,5'-Tetramethylbenzidine. Retrieved October 1, 2020 from https://pubchem.ncbi.nlm.nih.gov/compound/3_3_5_5_-Tetramethylbenzidine (2020).
  2. Magro, L. et al. Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics. Sci. Rep. 7, 1347 (2017).
  3. Cordray, M. S. & Richards-Kortum, R. R. A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA. Malar. J. 14, 472 (2015).
  4. Transcriptor One-Step RT-PCR Kit (50 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/roche/tosrtro?lang=en&region=NL
  5. KAPA PROBE FAST One-Step (20 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/roche/kk4752?lang=en&region=NL
  6. Titan One Tube RT-PCR System (25 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/roche/11855476001?lang=en&region=NL
  7. Quantitative RT-PCR ReadyMix™ (50 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/sigma/qr0200?lang=en&region=NL
  8. KiCqStart® One-Step Probe RT-qPCR ReadyMix™ (20 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/SIGMA/KCQS07?lang=en&region=NL
  9. SYBR® Green Quantitative RT-qPCR Kit (50 µL per reaction). Retrieved on October 3rd, 2020 from https://www.sigmaaldrich.com/catalog/product/sigma/qr0100?lang=en&region=NL
  10. SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (25 µL per reaction). Retrieved on October 3rd, 2020 from https://www.bioke.com/webshop/neb/e2019.html
  11. LavaLAMP™ DNA Master Mix for Amplification (25 µL per reaction). Retrieved on October 3rd, 2020 from https://www.lucigen.com/LavaLAMP-DNA-Master-Mix-loop-mediated-isothermal-amplification/
  12. TwistAmp exo RPA Kit (50 µL per reaction). Retrieved on October 3rd, 2020 from https://www.twistdx.co.uk/en/products/product/twistamp-exo

About Us

We are the 2020 team of iGEM Leiden. With an interdisciplinary team of students we aim to develop a point-of-care rapid diagnostic tool for infectious diseases!

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iGEM Team Leiden
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igem@science.leidenuniv.nl

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