Overview
This year, our project is expected to complete the examination of the hydatid patients.In the contribution section, our work are mainly divided into three parts:
1.Construct the plasmid containing the hydatid feature sequence.
2.Design multiple pairs of primers that could specifically amplify the hydatid gene fragment and explore the appropriate concentration for the amplification of the multiple primers.
3.Design multiple sgRNA that were matched to the amplified products based on the PAM sequences identified by crispr-Cas12a.
This laid the foundation for other teams to conduct future research on hydatid worms, and also contribute to the multiple primers amplification for other teams.
Plasmid (SE Plasmid)
The hydatid genes that can be detected in vitro are fragmented into fragments, and some fragments can be detected in most hydatidosis assays, and we constructed these characteristic sequences into plasmid and examined the corresponding fragments in subsequent experiments.
Primer
We designed 16 primer pairs for the characteristic fragments of Echinococcus and confirmed that 14 pairs can be used in amplification. Considering whether the corresponding fragments can design sgRNA and the amplification effect, we finally selected six effective primers to perform multiple primer amplification, and explore the stable system of multiple primer RPA amplification, namely to ensure a single each primer concentration of0.3 μ M.Other teams can refer to the multiple primer amplification.amplification.
Fig 2. Electrophoresis plots of SE plasmid amplified by different primers
The sgRNA and the reaction system
For the amplification products of the characteristic Echinococcus fragments, we designed 13 sgRNA, and the sgRNAs corresponding to mgs4, mgs5, mgs6, mgs9, mgs10 and mgs14 fragments were highly active for reference for other teams studying Echinococcus disease.
Fig 3. Cas12a detection using different sgRNA
