Team:Shanghai HS United/Results
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.
There are six essential components required by the reagent needed for the LAMP amplification: the DNA template, ddH2O, primers, dUTP, UDG, and a LAMP mixture (containing buffer, BST 2.0 enzyme, and dNTP). To reach optimum accuracy, sensitivity, and specificity for the detection, the most suitable primers and the amount of components must be selected first.
First, annealing short oligos (see figure feature) were used to mimic ASFv DNA target. Then, Cas12a detection reactions were performed by real-time fluorescent detector. We found the target-4 site had the best response speed and strength.
Under series of experiments and the final gel electrophoreses examination, primer LAMP-primer-4 finished amplification with higher quality compare to primer LAMP-primer-5 and with shorter time (30 mins), therefore primer LAMP-primer-4 is selected as primer used later on. (The sequence of primers, please see Design)
Throughout the course of the experiment, we have encountered one set of results displaying massive amount of successful amplification, even under conditions where amplification is not supposed to occur (when the template is ddH2O), which suggests that that round of experimentation might be contaminated by aerosol particles.
After adding the UDG to the original reagent and repeating the process of the experiment, the results under gel electrophoreses proves that UDG can effectively reduce potential contamination from the aerosol particles as the set of conditions with ddH2O as the template has failed to amplify. However, during our final experimentation, aerosol particles seem to have once again affected the amplification under the presence of UDG, therefore this is an issue we aim to resolve in the ultimate set of kit or product we design to make our detection method practical in real life.
Moreover, the most effective set of conditions is set with the template with the concentration gradient at 10-5, temperature at 65 ℃, and 30 minutes. Although other sets of condition have also presented effective results, one core feature of our way of detection is that it requires the minimum amount of time needed with accurate results, therefore the optimum set of conditions is determined with the concentration gradient at 10-5, temperature at 65℃, and 30 minutes.
Our iGEM team added ASFv DNA segments into the clean blood from pigs and diluted the samples into different concentrations. The diluted samples are tested under the same LAMP process and the results show that this amplification process still effectively amplifies samples with the concentration of 10-7nM.
To test our detection method’s specificity, the original targeted DNA template with ASFv was changed into two types of different bacteria DNA templates (Escherichia coli and Staphylococcus aureus). The results display by the test strips determine that the LAMP amplification method only proliferates the targeted ASFv templates, but not other bacteria DNA, proving that the method’s specificity solely on ASFv DNA is achieved.
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.
