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.

Why isothermal PCR?

Classical Polymerase Chain Reactions We chose to explore 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.

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 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. Activity could be shown on an agarose gel but first time stability experiments showed that the 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.
Furthermore, temperature seems to be a very important factor during amplification using RPA. As observable in Figure 2, the sample without any prior storage at a reaction temperature of 20 °C, though still active, showed dramatically decreased activity.

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 protein 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. The results are shown in Figure 3. A band at the expected length of the amplicon of 194 is visible. We observed that the yield measured by on-gel concentration determination was approximately the same.

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.