Throughout the project safety was always a priority when carrying out laboratory experiments and constructing our device. Since the beginning of the project, the team was aware of the large number of safety risks associated working with pathogens such as STEC E. coli, campylobacter, shigella and salmonella. No one on the team had experience working with or handling these nasty pathogens, so we had to take safety training very seriously and follow strict safety precautions.
Due to the health risks associated with these pathogens, the team underwent laboratory safety training on our first day in the lab. One of our graduate student advisors gave us an in-depth look at the laboratory’s safety features such as:
All team members had a general understanding of laboratory/workplace safety as we were already Workplace Hazardous Materials Information System (WHMIS) certified from pervious classes. Training with regards to laboratory procedures such as PCR and protein expression were carried out by our graduate student advisors Kody Klupt, Andrea Petkovic and Nolan Neville and PI Dr. John Allingham.
Before we got to testing actual water pathogens, we made it a priority to familiarize ourselves with LAMP. To ease our way up to LAMP, we first started with a standard PCR on a plasmid containing E. coli K12. Once we became familiarized with the lab space and became more experienced with laboratory equipment, we performed LAMP on extracted RNA from E. coli K12. To limit the amount of pathogen use, we also ran LAMP experiments with K12 to determine the optimal concentration of HNB dye. E. coli K12 does not normally colonize in the human intestine, survives poorly in the environment and is not known to have adverse effects on microorganism and plants. Due to its history of safe commercial use, we decided to use this strain of E.coli first.
Our initial hope was to test live water samples containing STEC E. coli, campylobacter, shigella and salmonella, however we quickly realized that these live pathogens required a level 2 lab space. After consulting with our PI, an alternative was to use heat killed pathogens as a substitute to directly test our LAMP primers. We were able to obtain lab strains of pathogenic E. coli from a lab and used it to test our LAMP primers. To take our safety concerns a step further, one of our graduate advisors performed an RNA extraction on the heat killed E.coli to ensure that it was only RNA we were working with and not the pathogen as a whole.
E. coli TOP10 and BL21 were the bacterium used for protein cloning and expression. These E. coli strains are not pathogenic, survive poorly in the environment and have a history of commercial use including pervious QGEM teams.
When performing all laboratory experiments, we ensured each team member wore proper personal protective equipment including lab coats, gloves, long pants, safety glasses and closed toe shoes. We were also sure to work in close proximity of our graduate advisors and PI.
Hydroxy Naphnol Blue (HNB) was the colorimetric dye used for our test. When consulting with its SDS, it was found that HNB can cause serious eye irritation if coming in contact with. Whenever handling HNB directly, we used PPE, eyewear and laboratory coats as means of protection.
Since our device is “kettle-like”, the most obvious concern was physical contact with the hot surface. To address this issue, we developed a custom insulative sleeve made which will house the device allowing for the user to pick it up without directly contacting the hot surface.
We also had concerns with the disposal of the LAMP test, as each individual test is a one-time reaction. We would plan on providing small hazardous waste bags to dispose of the LAMP reagents and potentially contaminated water. We acknowledge there is a possibility that the bags could break leading to a spillage in the environment. The amount of reagent in the LAMP cartridge is estimated to only be 50 microliters, any spill into the environment can be considered a contamination.
Through our interviews with members from Canadian Indigenous communities, we found that many people shy away from such at home devices due to their complexity. We plan on including a comprehensive user’s manual to guide the user on how to use each component of the device and how to interpret results. We have also developed a portable casing to hold all of the device parts and strategically designed it such that the parts are placed in order of set up.
