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Amplification Methods
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Since real-world patient's sample contain only traces of pathogens, detection with any
kind of read-out system will be difficult without prior amplification of the detected substance.
After we saw that the detection limit of Cas13a is in the nM region, we had to tackle the problem
of low target concentrations in medical samples.
This is why we explored different amplification methods. Our main idea was to keep things simple,
transportable and stable by incorporating isothermal reactions on paper with lyophilized reaction mixes.
Therefore, we explored isothermal PCR methods coupled to In-Vitro Transcription .
The final idea was to be able to detect both DNA and RNA samples by using either a
Reverse Transcriptase coupled Recombinase Polymerase Amplification and subsequent
In-Vitro Transcription for RNA targets or leaving out the step of reverse transcription for DNA targets.
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Why isothermal PCR?
We chose to explore isothermal methods for signal amplification since isothermal techniques
come with the potential of simplified hardware design which would lead to a decreased production and development costs.
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Recombinase Polymerase Amplification (RPA)
The Recombinase Polymerase Amplification 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 them 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
and ran dummy PCR reactions using the TwistDx RPA Kit. We tested this with standard Biobrick primers VF2 and VR and ran
the provided test reaction of the TwistDx RPA Kit as a positive control. The results are shown in Figure 1.
This approach is connected to some risk, since it is not said that PCR primers will work just as fine in RPA reactions.
The affinity of Recombinase to the primers is important to get an efficient binding of the primers to the template DNA.
For this reason PCR primers tend to be longer and have less GC% than RPA primers in general.
In addition, the tested construct was approximately 1500 bp long, which is also quite far from RPA's optimum of 500 bp.
Nevertheless, we could show that product is formed, though only up to the size of 500 bp. After that, it seems that the
Polymerase falls off the strand and was not able to produce longer amplicons. This is why one can see small bands for
longer constructs, just with a much lower yield.
The next step was to bring the RPA reaction on paper. For this, we lyophilised 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.
This did work out fine but first time stability experiments showed that the activity decreases quickly.
Also in the user's manual, it is said that after breaking the seal one should use up the reaction mix in the next hour.
We could underline this statement since activity declined rapidly after as little as two hours and was non-existent after
24 hours.
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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 at 37 °C of Control by TwistDx and His6-TEV using VF2 and VR primers.
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Figure 3: 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.
Coupling RPA to In-Vitro Transcription
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 was necessary. For this, we developed
a reaction mix in bulk that would perform both steps at a time. This is achievable since both reaction happen at 37 °C.
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Bringing RPA and In-Vitro Transcription on paper
The final step is bringing all the prior tested work on paper and stabilise it on their so it could be sealed into
the PDMS chip developed by the Hardware Team to automatise the amplification process and be able to subsequently
detect on a paperstrip using Cas13a from a simulated real-life sample. For this we ran tests and it did seem that
the RPA-Tx on paper could have worked judged by the 15%-Urea-PAGE. The gel showed two bands around the size of the
construct of 200 bp, one larger in size and less intense and one smaller in size and more intense. This could quite
possibly be the couple of DNA sample and derived RNA sample. Nevertheless, Cas13a-activity based on these samples
could not be shown.
Benchmark construct for Cas13a
Since the T7 RNA Polymerase only binds double-stranded DNA, transcription would never work form the His6-TEV
construct we initially tested RPA on, because the amplicon was too long. 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 on paper.
Bringing RPA and In-Vitro Transcription on paper
The final step was bringing all the prior tested work on paper and stabilise it on there so it could be sealed
into the PDMS chip developed by the Hardware Team to automatise the amplification process and enable
subsequent detection on a paperstrip using Cas13a. For this we ran test experiment
and we showed that RPA-Tx on paper worked judged by the 15%-Urea-PAGE. The gel showed a band at the approximate size
of 130 bp of the Benchmark construct that increased in concentration during the RPA-Tx reaction.
This could quite possibly be the couple of DNA sample and derived RNA sample. Nevertheless,
Cas13a-activity based on these samples could not be shown.
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 the customer.
One issue that can still occur 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 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 bringing this on paper in a lysate environment
might prove to be more difficult.
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References
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