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 concentration signals. 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 .

Why isothermal PCR?

We chose to explore isothermal methods for signal amplification since isothermal techniques come with the potential of simplified hardware design. Furthermore, this would lead to a decline in production and development cost.

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. Hence, the first step in the development of an isothermal amplification method 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 our standard Sequencing primers. This approach is connected to some risk, since it is not said that PCR primers will work just as fine in RPA reactions. In general, PCR primers tend to be longer and have less GC% than RPA primers. 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 fragments.

RPA on paper

The next step was to bring the RPA reaction on paper. For this, we lyophilised the reaction mix 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 stability does not seem to be too great. 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.

RPA 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 and thus humidity. We could dramatically increase stability when covering the paperstrip in a plastic Petri dish and sealing it with Parafilm.