Week 1 - (Apr. 25th-May 1st)
Plasmids containing the hydatid sequences were constructed, and the first RPA amplification kits, primers, and sgRNA were ordered.
Week 2 - (May 2nd-May 8th)
Repeated practice of molecular cloning technology, during this period, the plasmid extraction concentration is low, low transformation efficiency, the extraction products are impure and other problems occurred.
After this, we optimized the relevant experimental conditions and exercised the experimental skills during this period to avoid future mistakes.
Week 3 - (May 9th-May 15th)
The plasmid containing hydatid sequence was amplified and tested by sequencing. The plasmid containing the E gene was simultaneously amplified and tested by sequencing for subsequent tool validation and system construction.
Fig 1. Electrophoresis plot of SE plasmid
Week 4 - (May 16th-May 22nd)
Performed primer verification and sequencing test for correctness.
Week 5 - (May 23rd-May 29th)
We first used RPA for amplification, and the electrophoretic bands were not even clearly visible.
Fig 2. Electrophoresis plots of SE plasmid amplified by RPA
The first reason for the suspected failure was as follows: the protein is more tightly bound to DNA in RPA than in PCR, and it remains as a complex in electrophoresis.
Improvement scheme: phenol-chloroform.
However, there is still the problem of unclear strip after the improvement.
Fig 3. Electrophoresis plots of purified SE plasmid amplified by RPA
Week 6 - (May 30th-Jun. 5th)
Initially we hypothesized that cas12a detection was more sensitive than agarose electrophoresis.
So plan firstExploring the conditions of cas12a shear, the first version of the reaction system was obtained, and after many repeats, stable results were obtained.
Week 7 - (Jun. 6th-Jun. 12th)
Failure of cas12a detection using the RPA amplification products.
After repeated RPA several times without ideal results, we suspected that the RPA kit was problematic. After consulting with teachers with a relevant research background, a new RPA kit was ordered.
Week 8 - (Jun. 13th-Jun. 19th)
In the early test, the colloidal gold test paper appeared an unstable problem, and we will focus on solving it this week.
The results showed that this batch of test strips had serious quality control problems, and we decided to order products from other manufacturers.
Week 9-Week 11 - (Jun. 20th-Jul. 10th)
Review and prepare for the final exam, and participate in the field internship organized by the college.
New RPA kit and test strips arrived.
Week 12 - (Jul. 11th-Jul. 17th)
Reamplify the hydatid sequence-containing plasmid, and test the plasmid sequence correctness by sequencing to ensure the plasmid quality.
Test the quality of the new test stripe, and get a reliable reaction system and probe carrying capacity.
Fig 4. Testing the test strip by different concentration of reporters
Week 13 - (Jul. 18th-Jul. 24th)
RPA amplification and cas12a detection were performed with the E gene as the template result.
RPA amplification products were tested for the minimum detected concentration.
Fig 5. Exploring the Cas12a detection limit using E gene
To reduce the cost, we used fluorescent probes instead of colloidal gold test strips before formal testing, and determined the fluorescent probe use concentration.
Fig 6. Exploring the fluorescent reporter concentration
Week 14 - (Jul. 25th-Jul. 31st)
Hydatid sequence primer testing using RPA to remove inefficient primer pairs. Test results are repeated inspection to ensure authenticity.
Fig 7. Electrophoresis plots of SE plasmids amplified by RPA using different primers
Week 15 - (Aug. 1st-Aug. 7th)
We explored the multi-pair primer RPA reaction system and achieved stable multi-primer amplification.
Week 16 - (Aug. 8th-Aug. 14th)
Multiple sgRNA-guided cas12a cleavage after single primer amplification was tested using a hydatid-containing sequence plasmid, yielding stable results and removing inefficient sgRNA.
Fig 8. Determining the result of Cas12a detection using different sgRNA
Week 17 - (Aug. 15th-Aug. 21st)
Multiple sgRNA-guided cas12a cleavage after multiple primer amplification was achieved using hydatid-containing sequence plasmids, which showed that multiple sgRNA guidance has a higher activity in our project compared to single sgRNA guidance.
Week 18 - (Aug. 22nd-Aug. 28th)
Multiple sgRNA-guided cas12a detection after polyprimer RPA amplification was achieved using a synthetic DNA mimicking the hydatid disease cfDNA.
Week 19 - (Aug. 29th-Sept. 4th)
Results Use colloidal gold strips to report the multi-sgRNA-guided cas12a shear results of synthetic DNA that mimic the D N A of hydatid cfDNA after multiprimer RPA amplification.
Fig 9. Determining the result of the test strip
Week 20 - (Sept. 5th-Sept. 11th)
The synthetic DNA samples, including the cfDNA, the simulated schistosomiasis DNA and the simulated human genome, were used to calculate the true positive rate and the false positive rate.
The positive samples were also subjected to a single sgRNA-guided cas12a test after a single primer to review the sequences present.
Week 21 - (Sept. 12th-Sept. 18th)
Organize the experimental results and plot the results.
