The system construction of POIROT can be broadly divided into four parts: biomarker, amplification, detection, and visualization (Lateral Flow Assay, LFA). In particular, since there are various methods for amplification, we conducted many experiments.
- We were able to demonstrate that TWJ-SDA has significantly superior specificity compared to NJ-SDA.
- Using a uniquely designed let-7b single nucleotide variant, we evaluated the sequence specificity of TWJ-SDA and confirmed that the specificity changes depending on the position of the mutation. This allowed us to establish guidelines for the appropriate design of helpers and templates for new biomarkers.
- We were able to connect TWJ-SDA and multistep-SDA. Among these, we successfully achieved detection at the fM order with TWJ-SDA > 3step-SDA > MB. Additionally, we also succeeded in detecting at the fM order when the target miRNA was diluted with artificial tears instead of TE.
- We successfully connected multistep-SDA and Cas3. Notably, we achieved detection down to 1 fM with the combination of 3step-SDA > ds Amp > Cas3.
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All achievement above was conducted Isothermally, at 37 ℃.
To our knowledge, there have been no previous examples of connecting TWJ-SDA with multistep-SDA or multistep-SDA with Cas3. Therefore, we can confidently say that we have independently developed a detection system that has both high selectivity and sensitivity.