Most of our lab work was completed by Mercy Awosoga this summer as part of the High School Youth Researcher Summer program at the U of L. This is a six week intensive research program for students in grade 11. They are paired with a lab at the U of L to work on a research project during the summer. Mercy was supervised by our Faculty Advisor Dr Laura Keffer-Wilkes. She was able to complete a lot of wet lab work as well as a lot of the hardware engineering. Many thanks to Mercy for all her hard work!
RPA vs PCR Detection of Purified Plasmid
We confirmed that the primers we designed for RPA work as intended using purified plasmid DNA. The primers should produce products of 427 bp and 585 bp, respectively from the asPink and GFP DNA templates. (Figure 1).
Figure 1. Plasmid maps of pSB1C3-asPink (left) and pSB1C3-GFP (right) used as template DNA in our proof-of-concept reactions. Primer annealing regions are shown in purple.
We were successful in producing the right size product for both asPink and GFP templates using RPA but did not amplify asPink using PCR. Overall, our primers were successfully used in RPA with a purified plasmid template (Figure 2).
Figure 2. Analysis of PCR and RPA proof-of-concept reactions using 1% agarose gel.
DNA Extraction using Boiling Technique
The two proof-of-concept cell strains carrying pSB1C3-GFP and pSB1C3-asPink were grown in LB overnight. Cells were collected by centrifugation and then resuspended in fresh LB. Into a new tube containing PBS, 1 OD of each cell strain was added. This was repeated three times for a total of four sample tubes each containing an equal number of cell strains. The tubes were then subjected to heating at 100C for 0, 2, 10, or 20 minutes. A 100 uL sample was removed from the boiled tubes and plated on LB plates and grown overnight (figure 3). No growth was seen on plates with samples that had been boiled for 10 or 20 minutes but some growth was observed at the 2 minute time point allowing for quantification of the different cell strains, which was approximately 50:50. A lawn of bacterial growth was observed on the 0 minute boiling time point, which was expected.
Figure 3. An aliquot of the boiled cell solution was plated on LB and grown overnight. Growth was only observed on the no boil and 2 min boil plates.
Next, the boiled solutions were used as the DNA template for RPA reactions. This would demonstrate that enough DNA is present following the boiling step for DNA detection in the field. We were able to detect the GFP plasmid even with no boiling, indicating that perhaps during our preparation steps some of the cells are bursting without the need to boil. We were also able to detect purified templates as low as 2.5 ng.
Figure 4. The pSB1C3-GFP plasmid was able to be detected in all samples, even at 0 min boiled.
Unfortunately, we had more trouble being able to detect the asPink plasmid in the boiled cell samples (below). We have to do some troubleshooting for this detection step.
Figure 5. The pSB1C3-asPink plasmid was only detected in the 10 and 20 minute boiled samples. No DNA amplification was observed for any of the BRD pathogen DNA.
We also need to troubleshoot detection of the BRD pathogen DNA. Dr Tim McAllister and Cheyenne Conrad, both from the Lethbridge Agriculture and Agri-Food Canada Research Station, provided us with purified DNA from the four main BRD pathogens, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. This allowed us to test our RPA pipeline but still remain safe. Unfortunately, we were unable to detect the DNA using our primers and RPA components (figure 5 lanes 6 - 9). Using a higher percentage agarose gel may help with resolution for detection.
Hardware and HNB Testing
We were able to test our hardware prototype in the lab. It was able to heat the RPA reactions to 37℃ (figure 6)
Figure 6. Bo-Find prototype heating RPA samples.
Interestingly, when we used HNB as our detection method, we observed a colour change despite not being able to detect a band on the gel for the asPink sample (figure 7).
Figure 7. Analysis of RPA proof-of-concept reactions using 1% agarose gel (right). HNB was also used to detect DNA amplification (left).
Summary
We were able to successfully use RPA to detect GFP and asPink plasmids from purified DNA but more troubleshooting is needed for confirmation and accuracy. DNA extraction via boiling demonstrated that DNA could be detected in as little as 2 minutes, meaning no elaborate extraction method is needed in the field. BRD pathogen DNA detection was unsuccessful with current RPA components, therefore more fine tuning needs to be completed before field deployment, Despite the correct colour change being observed with HNB, no band on the gel was observed indicating a need for further optimization. The hardware prototype effectively maintained the RPA reaction temperature at 37°C.
