Through repeated experiments, we have independently developed a detection system that has both high selectivity and sensitivity. However, further experiments, as outlined below, are necessary to complete POIROT.
- We will verify whether we can amplify the target miRNA in actual tear fluid, which is a complex environment with many contaminants. First, we will amplify miRNAs that are known to be relatively abundant in tear fluid of the general population to confirm that our developed system works in human tear fluid.
- We evaluated the sequence specificity of TWJ-SDA and established guidelines for the design of appropriate helpers and templates. By utilizing this, we will design helpers and templates for various miRNA sequences and conduct experiments, ultimately demonstrating that POIROT can be used for the amplification and quantification of miRNAs with all types of sequences.
- With TWJ-SDA > 3step-SDA > ds Amp > Cas3, we were able to distinguish between a target concentration of 100 fM and 0 M. However, when the target concentration exceeded 1 pM, the signal became obscured, making it impossible to differentiate. By optimizing template concentration, polymerase concentration, and nickase concentration, we anticipate being able to construct a system applicable to a broader range of target concentrations.
- With the 3step-SDA > ds Amp > Cas3 system, we successfully achieved detection down to 1 fM. However, this system currently lacks quantitativeness, so we aim to establish quantification using FQ probes by optimizing template concentration, polymerase concentration, and nickase concentration.
- In our experiments using commercial LFA products, adjusting the viscosity of the buffer proved challenging, resulting in instances where the T-line showed color under conditions that should be negative, or the T-line did not show color under conditions that should be positive. Developing a strip independently is difficult for us, so we aim to create a POIROT-optimized strip by collaborating with companies that already have the necessary expertise.
To complete POIROT, which enables the amplification and quantification of miRNAs with any sequence, we will continue our experiments.