The Significance of Modeling
Modeling has the following advantages
The results can be estimated and predicted before the laboratory experiments are performed.
It can improve the efficiency of experiments by prioritizing experiments that are expected to produce good results.
The possibility of further improvement of the experiment can be expected by the good results suggested by the modeling.
We have considered the advantages above in predicting the time required for virus detection using a physical model
We have considered the advantages above in predicting the time required for virus detection using a physical model
Method
We used the method of iGEM Munich2017 as a reference. The chemical reaction equations and differential equations used are as follows.
In order to evaluate the rapidity of virus quantification, we simulated the transition of the fluorescence intensity of the device. The parameters used were as follows.
In order to evaluate the rapidity of virus quantification, we simulated the transition of the fluorescence intensity of the device. The parameters used were as follows.
Result
The results of the modeling using the above chemical reaction equations, differential equations and parameters are as shown in figure 1.
Figure1. Result of our modeling
We used 1 nM of Cas12a, 10 nM of crRNA, and various concentrations of target. The target RNA at 10 nM and 1.0 nM was almost completely cleaved in about 5 minutes. Even 0.1 nM target RNA showed detectable cleavage activity in about 30 minutes.
Figure1. Result of our modeling
We used 1 nM of Cas12a, 10 nM of crRNA, and various concentrations of target. The target RNA at 10 nM and 1.0 nM was almost completely cleaved in about 5 minutes. Even 0.1 nM target RNA showed detectable cleavage activity in about 30 minutes.
Expectations for the future
The results of our modeling suggest that our HHV-6 quantification device may be able to quantify human herpesvirus in about 5 minutes of reaction time. In contrast, the conventional quantification method requires about 1 hour and 30 minutes of reaction time due to the use of the real-time PCR method. The HHV-6 quantification device we developed is simple and rapid, and is expected to be widely and routinely used as a simple method for the quantification of fatigue.
References
1) Nalefski, E. A., Patel, N., Leung, P. J. Y., Islam, Z., Kooistra, R. M., Parikh, I., Marion, E., Knott, G. J., Doudna, J. A., le Ny, A.-L. M., & Madan, D. (2021). Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics. IScience, 24(9), 102996.
2) Strohkendl, I., Saifuddin, F. A., Rybarski, J. R., Finkelstein, I. J., & Russell, R. (2018). Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Molecular Cell, 71(5), 816-824.e3.