Team:Shanghai HS United/Demonstrate
To achieve the ultimate goal to reach optimal simplicity, immediacy, and accuracy, our iGEM team decides to use the LAMP + Cas12a + lateral flow method to amplify potential ASFv-affected genetic segments from the pig population. The final results of our wet-lab experiments show that our attempts on improving the current detection method is feasible.
To examine the Cas12a reaction, we added crRNA, FnCas12a, the LAMP products, FITC-N14-Biotin, 10×NEB Buffer 3.1, and ddH2O to create a 20 μl reagent.
After reacting under 37 ℃ for 10 minutes, the reagent is tested by the fluorometer. From the results, Cas12a can detect concentration similar to that detected directly by the LAMP fluorescent — 10-7 nM.
Base on the LAMP product above, we take a control and templates with concentration of 10-7 nM and conduct the same Cas12a reaction. After the reaction, we add buffer MGCB into the reagent and insert the HybriDetect dipstick (test strip). As the results, the blood samples containing designed ASFv DNA segments display 2 strips on the dipstick, meaning a positive result proving the sample is affected by the ASFv.
We tested all above LAMP products (gel electrophoresis samples) for lateral flow test, it showed the same results.
The results of our experiments prove that the sensibility, specificity, and rapidity we desire is reached. However, the accuracy we aim to achieve still needs future improvements to be made on the product we design.
Our iGEM team designed a model of our future product that can be put into use in real life. The kit contains the mixture of reagents without the template, which would be added in by the pig owners with the samples from the pigs’ blood or fodder. To maximize the reduction of contamination from aerosol particles, we wish to create the kit to be completely enclosed since the environment and personnels operating the detection might not be necessarily professional or well-prepared.
