Introduction
The kit designed by our project is the first experimental scheme to achieve device-free multi-primer RPA amplification and multi-sgRNA Cas digestion since 2020.
With full reference to the original literature system, we fail to amplify the expected sequence, so we continuously explore the system, proportion and temperature, reaction time and ask medical workers doing nucleic acid testing in actual application according to the system and developing the incubation method for amplification. Verified by multiple tests, this method can obviously improve the success rate of RPA and accuracy, the expected results accord with experiment. It also has a significant effect on subsequent CRISPR-CAS cutting.
After ensuring the success rate of RPA and CRISPR, we verified the effectiveness of the primers and sgRNA. Finally, 6 pairs of correct and efficient primers and 6 suitable sgRNA were screened. Due to the mismatching of stem-loop structure and incomplete adaptation of PAM sequence, we excluded two sgRNA, improved them by further literature review, and redesigned them so that they could be used normally.
Fig 1. Cas12a detection using different sgRNA
At the beginning, we selected reporters of 5 nt and 8 nt according to the literature, but it was found in the actual experiment that the binding efficiency of these two reporters was not high. Therefore, we designed a 15 nt reporters according to the characteristics of cfDNA and the target sequence after discussion, and the comparison results showed that the reporters was significantly better than 5 nt and 8 nt.
Fig 2. Cas12a and fluorescence detection using different concentration of reporters
