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Results: Amplification
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Since real-world patient samples contain only traces of pathogens (attomolar at the lowest), any detection method relying on binding affinity (such as Cas13a with target RNA) needs prior amplification of the detected molecule to concentrations within the range of the Kd.
After we confirmed that the detection limit of Cas13a is in the nanomolar region, we had to tackle the problem
of low target concentrations in medical samples.
We explored different amplification methods that would be simple and stable on our transportable platform. We wanted to incorporate isothermal reactions on paper with lyophilized reaction mixes.
Therefore, we explored isothermal PCR methods coupled to in-vitro transcription . We chose isothermal methods for signal amplification since isothermal techniques
come with the potential of simplified hardware design which would lead to decreased production and development costs. PCR using a proper thermocycler and well-defined cycling times also require a trained personnel to use them, which does not fit to our goal of a distributable diagnosis device.
Eventually, we would need to detect both DNA and RNA samples by using either a
reverse transcriptase coupled isothermal recombinase polymerase amplification and subsequent
in-vitro transcription for RNA targets or leaving out the step of reverse transcription for DNA targets. We term this amplification process RT-RPA-TX.
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Recombinase Polymerase Amplification
The Recombinase Polymerase Amplification (RPA) developed by TwistDx is an isothermal amplification method for DNA.
Rather than melting the double strand and annealing the primers through temperature cycles, it uses a recombinase
that binds the primers and assists in the annealing process. Another protein, single-strand DNA binding protein (SSB)
promotes the binding of the primers to the recombinase in this process.
Hence, the first step in the development of an isothermal amplification method of an RNA signal via RT-RPA-TX was testing the
Recombinase Polymerase Amplification (RPA) itself in bulk. For this, we took our previously cloned His-tagged TEV Protease (BBa_K2323002 improved from BBa_K1319008)
and ran dummy PCR reactions using the TwistDx RPA Kit and the universal biobricks primers VF2 (BBa_G00100) and VR (BBa_G00101). We ran
the provided test reaction of the TwistDx RPA Kit as a positive control (TwistAmp exo). The results are shown in Figure 1.
The first point to note is that PCR primers and RPA primers have different requirements, due to the need for affinity of the recombinase to the primers (RPA primers tend to be shorter with a higher GC content). Therefore VF2 and VR might not be ideal primers for RPA. The second point is that RPA's optimum amplification length is 500bp. For longer amplifications, it is possible that the polymerase falls off the strand. This could explain why we see at maximum a band of 500bp, and shorter bands, although our amplicon should be 1262bp. We could therefore see that amplification using RPA was possible in bulk, but that primer design would need to be optimized for our final product.
RPA on paper
The next step was to bring the RPA reaction on paper. For this, we lyophilized the reaction mixture provided by TwistDx
on paper and tried to run RPA reactions directly from it by inserting the blotting paper into the PCR tube.
Activity could be shown on an agarose gel but time-stability experiments showed that the RPA kit activity decreases quickly.
In the user's manual, it is stated that after breaking the seal, one should use up the reaction mix in the next hour.
We can confirm this statement since activity declined rapidly after as little as two hours and was non-existent after
24 hours, as is visible in Figure 2.
Furthermore, temperature seems to be a very important factor during amplification using RPA. The samples incubated at of 20 °C, though still active, showed dramatically decreased activity compared to the same samples at 37°C.
For usage of RPA on a detection device that does not require purification of the sample, the reaction needs to take
place in the environment of a lysate with cell debris and released proteins from the cell. In order to see whether this
is achievable, we performed a colony-PCR like setting of RPA using a E. coli culture in LB-medium with OD=1.0
and ran a normal RPA of purified plasmid as a control. As our first PCR produced too long of an amplicon, we changed to a shorter PCR scheme, which should lead to an amplification of 194bp. The results are shown in Figure 3.
A band at the expected length is visible in both the control condition and the E.coli environment. We observed that the yield measured by gel quantification was approximately the same.
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Figure 1: RPA reaction at 37 °C of Control by TwistDx and His6-TEV using VF2 and VR primers.
Figure 2: RPA reaction of lyophilised RPA mixture at 37 °C or 20 °C of His6-TEV using VF2 and VR primers.
The control is a reaction mixture without the lyophilisation step.
Figure 3: Colony-PCR of Cas13a Benchmark plasmid using RPA. Control is a standard RPA reaction of purified plasmid
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Figure 4: RPA reaction after freeze-dried storage on paper at different conditions. Conditions that were taken
into consideration are temperature and air accessibility. Air determines the samples that were accessible to air.
The other samples were stored in a Petri dish sealed with parafilm.
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RPA on paper time-stability optimisation
Since stability is an important question when developing a diagnostic test, we examined the bottleneck to the stability of
RPA reaction mix on paper. Basically, there is only two possible, though obvious, factors affecting the stability:
Exposure to humidity and temperature. So we tested both of these factors in an experiment and found out, that the bottleneck
is mainly presented by exposure to air, presumably humidity. We could dramatically increase stability when covering the
paperstrip in a plastic Petri dish and sealing it with Parafilm. The results are shown in Figure 3.
Benchmark construct for Cas13a
The fact that Cas13a is a RNA-guided RNAse made it necessary to not only amplify a DNA signal but also transcribe
it into RNA. Thus, coupling the RPA reaction to In-Vitro Transcription (RPA-Tx)was necessary.
For this, we needed to developede
a reaction mix in bulk that would perform both steps at a time. This is in theory achievable since both reaction take place
at a temperature of 37 °C.
We thus proceeded to try RPA-Tx on the His6-TEV construct but we never succeeded to get the expected bands in
the Urea-PAGE after Phenol/Chloroform extraction. After some revision we realised that, since the T7 RNA Polymerase
only binds double-stranded DNA, transcription would never work from the His6-TEV
construct we initially tested RPA on, because the amplicon was too long for RPA to form the full double stranded amplicon.
An option would have been to order a different
primer. It comes with the risk of losing RPA activity due to the strict dependency on the primer mentioned above and would
need additional time for optimization. Thus, we decided against that option and constructed a benchmark target RNA plasmid for
Cas13a. This construct consists of target sequences we took for 16s rRNA of E. Coli , 16s rRNA of B. subtilis
and the 5'-UTR of the norovirus. It is flanked by VF2 and VR. Upstream of the target sequences is a T7 promoter that allows
In-Vitro Transcription. After cloning this into a plasmid, we had a system with which we could test our coupled RPA-Tx
and check whether it works in general and on paper.
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Bringing RPA and In-Vitro Transcription on paper
The final step was stabilizing all the prior tested work on paper and stabilise it on there so it could be sealed
into the sample processing unit developed
by the Hardware Team to automatise the amplification process and enable
subsequent detection on a paperstrip using Cas13a. For this we performed experiments
and showed that RPA-Tx lyophilised on paper worked judged by the 15%-Urea-PAGE. The gel for RNA qunatification is shown in Figure 5.
The gel showed a band at the approximate size of 130 bp of the Benchmark construct transcript that
increased in concentration during the RPA-Tx reaction.
We were able to perform the experiment in a time dependent measurement ending the RPA-Tx reaction after 30 minutes,
60 minutes and 2 hours. The experiment suggested that the transcription of DNA template into RNA is mainly restricted by
the speed of the T7 RNA Polymerase since the reaction speed seemed to be constant over the observation time. The concentration
of RNA at overnight transcription was not measurable since the gel was completely overloaded which is most probably a result
of incomplete DNase I digestion after the transcription due to immense amount of DNA after running the RPA this long.
Finally, we proceeded to test the ability of our product from RPA-Tx to trigger Cas13a collateral RNase activity.
Due to time limitations, we were not able to show time stability of the RPA-Tx mixture anymore.
Figure 1: RPA reaction at 37 °C of Control by TwistDx and His6-TEV using VF2 and VR primers.
Discussion
We showed here the first steps of establishing an amplification circuit for our detection device.
Since we were able to screen for storage conditions and do colony PCR-like experiments with the RPA mixture on paper,
the reproducibility of the RPA reaction after dry-freezing it on paper is proven. Time-stability has
also been shown for 24 hours, though this will need to be extended to longer time-scales to provide
it to customers.
One issue that can still needs to be solved is that so far, the coupled RPA-Tx reaction was only tested for purified samples.
The fact of having lysate in the sample is likely to have distorting effects on the efficiency on both reactions.
Since RPA worked in a colony PCR-like set up with prior lysis of the cells, that is already a good start but the
In-Vitro Transcription is usually more delicate, therefore performing it in a lysate environment
might prove to be more difficult.
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References
- Gao, Zhu and Huang. "Recombinase Polymerase Amplification: A New DNA/RNA Amplification Strategy." (2016)
Chinese journal of Biochemistry and Molecular Biology 32(6): 627-634.
- Daher, Stewart, Boissinot and Bergeron. "Recombinase Polymerase Amplification for Diagnostic Applications"
(2016) Clinical Chemistry 62(7): 947-958.
- Beckert & Masquida "Synthesis of RNA by In Vitro Transcription." Methods in Molecular Biology
(2010) Volume 703
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