Detection Limit
One major concern when dealing with the problem of diagnostics on patients is extracting the sample with which
detection can actually be performed. Since we wanted our method to be non-invasive, one concern that we needed to
deal with is the concentration of pathogens and thus detectable RNA in the patients mucus or other non-invasive sample. First approximations from different papers already showed that virological samples show concentrations no higher than low pM and can even go as low as aM.
Our wetlab experiments indicated that the detection limit of the Cas13a RNase activity is in the range of 10 nM.
Using our kinetic data, we estimated the rate constants for the different reactions to create a simple ODE model.
The chemical and differential equations for the model are shown below:
As shown in Figure 1, our simulations are able to reproduce the behavior observed experimentally.
Figure 1: Kinetics of Cas13a using 1 nM Cas13a and 10 nM crRNA at different target concentrations.
Next, we analysed the amount of readout RNA that was cleaved after 30 minutes for varying target concentrations. As shown in Figure 2, the curve follows a sigmoidal behavior and suggests a detection limit in the range of 10 nM. Due to this result, our initial design of applying the lysed and purified RNA sample directly on the detection paper strip had to be discarded. Since it is known from literature that Cas proteins show activity independent of their activation mechanism at high concentrations, we could not increase the concentration of Cas13a to improve the sensitivity. Instead, we explored amplification methods upstream in the detection process.
Figure 2: Estimated detection limit determined for the Cas13a system using 1 nM Cas13a and 10 nM crRNA.
Improved Reaction Cascade
In collaboration with our wetlab team we developed a reaction cascade for sample pre-amplification by coupling reverse transcription to isothermal recombinase polymerase amplification and transcription (RT-RPA-TX), resulting in auto-catalysis of target RNA (Figure 3).
Figure 3: Scheme for the RT-RPA-TX amplification system.
In order to compare the detection limit of the Cas13a system alone with the detection limit of the amplified the reaction cascade, we expanded our model, assuming exponential amplification of the target RNA. As the amplification reaction saturates due to a depletion of resources, the amplification stops as soon as the target RNA level reaches an upper limit of 1000 nM (Figure 4).
Figure 4: Schematic representation of the target RNA amplification during the estimation of the detection limit using the reaction cascade.
The kinetics for the amplfication cascade coupled to Cas13a based detection are shown in Figure 5. Strikingly, the start of the reaction seems to be determined by the amplificaiton reaciton, while the consecutive phase is limited by the rate of Cas13a mediated cleavage.
As shown in Figure 2, the detection limit of the reaction cascade decreases by approximately three orders of magnitude. These simulations led us to implement our pre-amplification cascade into our CascAID system.
Figure 5: Kinetics of the Cas13a systemusing 1 nM Cas13a and 10 nM crRNA at different target concentrations using the reaction cascade.
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