As of now there are very few reliable water pathogen testing devices on the market for point of care use. This is because there are so many possible pathogens in drinking water with varying physical differences as well as low concentrations of pathogens in water samples which are difficult to detect (1). The most important requirements for reliable analysis include: specificity, sensitivity, reproducibility of results, speed, automation and low cost (2). Typical pathogen detection in water is carried out in a lab using culture dependent methods, however these methods are limited by their low sensitivity and excessive time needed to obtain results (3). Our project this year hopes to bridge the gap between analysis specificity, reliability, time for results and overall cost centering around an emerging Nucleic-Acid-Based Method called Loop Mediated Isothermal Amplification (LAMP). We hope our method can be widely applied and useful for our direct stakeholders.

The intended end-user for our device is Canadian Indigenous people without clean drinking water and our device has been designed and iterated mostly based on meetings and feedback from Canadian Indigenous peoples. Throughout the duration of our project, we have also identified three other groups of potential end-users that could benefit from our device and met with these stakeholders to learn how our idea could help them. These other groups are: Canadian Farmers/Rural communities, outdoor enthusiasts, and Academia.

Most of the device was designed based on the meetings we had with Canadian First Nations peoples and water quality experts in the field. We learned that many of these people want to know what exactly is in their water and what was causing them to get sick. Many communities’ resort to shipping water bottles in which can become costly or use water trucks which carry all sorts of contaminants. Locals typically boil water for purification or use chemicals such as Chlorine. We designed a device that could filter out heavy metals through mechanical filtration, purify through boiling whilst testing for harmful pathogens in water. Our solution was confirmed and refined through various meetings with Canadian Indigenous Canadians such as members of the Queen’s Office of Indigenous Initiatives, the Chief of Tyendinaga (closest Indigenous community to Kingston).

A major hurdle for previous POC water devices was the complexity of use, especially for older individuals. Thus, we attempted to design an all-in-one device which incorporates the water test and purification within a few movable pieces. We decided to use a colorimetric indicator as this would not require further expensive hardware such as a flourometer and suggest Lyophilization of the reagents such that they can be stored at room temperature (4).

We see our device implemented in two different ways in these communities:

1. A device per household.
2. A device(s) in local community centers where residents can bring water samples to get tested from there. A few members of the community could get trained on how to operate the device and interpret results, thus would make our device more feasible for older residents.

Another group that may benefit from our device would be Canadian Farmers or non-Indigenous Canadians living in rural communities. Many rural communities face similar water quality issues as Canadian Indigenous communities as a lot of these communities suffer from water drainage from farmland containing harmful bacteria, parasites, viruses and metals (5).

Water quality is always a concern when raising animals on a farm or growing crops, however it was reported that only 7% of Canadian Farmers reported testing twice a year and over 64% did not test on a regular basis (6). Pathogens in water could infect or be carried by livestock to humans or other animals resulting in pathogenic outbreaks.

Our device could also be used by Outdoor enthusiasts such as campers to purify and test water. After speaking with an avid camper, we learned that water purification is typically done using purification tablets, however boiling is still the most effective technique (7). There are some pH, metal and coliform kits on the market, however nothing specific to testing for individual pathogens. Outdoor enthusiasts would prefer a device that has a minimal weight and is small enough to carry in a backpack for water purification and testing. We adapted our device such that it could be portable through the design of a built-in heat plate and added a third lid from which the user could drink directly from the device.

LAMP is an emerging Nucleic-Acid-Based Method, most recently with notable work in the detection of SARS COVID-19 (8-10) and commercial testing kits from companies such as New England Biolabs© and Lucira©. A number of researchers demonstrated LAMP as the most effective tool for coronavirus infection (11), similarly LAMP has shown the potential to detect a number of other viruses such as adenovirus (12), influenza A (13), herpes (14) and cytomegalovirus (15).

To contribute to future work, we wrote an article for the MSP-Vector iGEM journal in collaboration with the MSP-Maastricht iGEM team. We documented our fusion protien of a mutagenetic Bst version and DNA-BP Sac7e to optimize the reaction through the polymerases enhanced processivity and thermostability. We also constructed a 3D printed prototype of our device. With our journal article and prototype, we hope that future academics or iGEM teams could build off our project.

Our Bst construct was designed for universal use and HNB dye only relies on pyrophosphate generation, thus the only moving piece in our reaction are the primers. Following a standard procedure below, a new set of LAMP primers can be developed to test for other pathogens/microorganisms/viruses in water and implemented for our device.

After consulting an expert with a water diagnostic test on the market we learned about the steps we would need to take to produce a reliable drinking water diagnostic test. The main takeaway was for such water pathogen diagnostic test to be implemented on the market, the detection limit must be able to detect a single E. coli cell in 100 mL. Achieving this detection limit accompanied with device prototyping and testing takes years, thus with the creation of our fusion protien we hope to lower the detection limit of our LAMP assay to get closer to this gold standard detection limit. The general steps to get a product as such on the market is documented below:

1. Proof of concept on laboratory samples. Ensuring the origional method of detection works on cells cultured in the lab under controlled conditions.
2. Proof of concept on real world samples. Growing and culturing cells from external sources and ensuring the detection method works.
3. Prototype or “pilot” stage. Constructing multiple versions of the prototype that give reproducible results. The prototypes are integrated with some commercial parts with some non-commercial parts. This stage shows that the product is feasible, however is not commercial ready as of yet due to the cost. Cost takes into account laboratory work, reagents, time, salaries etc.
4. Consult with manufactures. Once you are nearing the end of the pilot phase, you will have to consult with manufactures to negotiate a reasonable cost to produce the device.
5. Approval from regulators. In Canada, we would have to get approval from the Ministry of Environment Drinking water division or approval from the United States Environmental Protection Agency (USEPA) which is globally recognized as valid approval. These regulators contract out to a 3rd party lab which follow a specific protocol to ensure the device will work. You do not need regulation to test sewage water, however you do need a regulation to test for sewage effluent, lake water, public water and drinking water.

Obviously, the limit of detection for any new laboratory tests will not be able to detect a single E.coli cell in 100 mL of water. To get to this stage there are mini milestones we aim to achieve such that are test can be certified for other applications before it gets to drinking water:

1. Successful detection on non-pathogenic lab E. coli strains
2. Successful detection on heat killed pathogens we are aiming to test
3. Expression and purification of fusion protien
4. Successful detection on non-pathogenic lab E. coli strains using fusion protien.
5. Successful detection on heat killed pathogens using fusion protien
6. Successful detection on diluted samples of cells using fusion to confirm lower detection limit
7. Successful detection on sewage water – first non-regulatory test
8. Successful detection on sewage effluent – must detect 200 CFU per 100 mL.
9. Successful detection on lake water – must detect under 200 CFU per 100 mL.
10. Successful detection on drinking water – must detect 1 E. coli cell per 100 mL.

We also learned that non-culturable parasites such as cryptosporidium and giardia are non-culturable, thus are extremely difficult to detect in the lab. Typical testing for these parasites involves bolting a test cartridge onto a pipe and running water through it to capture these parasites. The water must be run for at least two days to ensure a sufficient amount has been captured. We can envision our device also testing for these parasites as all we require is the 16s rRNA without any lab culturing.

Since the LAMP reaction will require a temperature of at least 65˚C, it will require a heating component. Since we have implemented a “kettle-like” device, our immediate concern was physical contact with the hot surface. We have developed a custom insulative sleeve which will house the device and allow the user to hold the device. A further step to ensure safety would be to incorporate a handle on the device to ensure the user is not making any contact with the boiling water compartment. A handle may also make the device easier to carry.

We also acknowledge that the colorimetric indicator dye we hope to use can cause eye irritation. However, we found this dye to be a much safer alternative to pH sensitive dye Phenol red which is used in commercial SARS COVID-19 LAMP kits. Phenol red can cause skin irritation, eye damage and organ toxicity. To ensure maximum safety, a solution could be to lyophilize all the reagents together including the dye to ensure the user does not make any contact with the mixture. We also plan on including biohazard bags to discard the reaction tubes and prevent any spillage.

To implement our system into these communities there are still some challenges that need to be addressed and considered.

The most obvious challenge is optimizing LAMP to detect 1 E.coli cell in 100 mL. This will involve extensive R&D and many trials of reaction optimization. To get even closer to this limit, further modifications may need to be made to our polymerase to increase its sensitivity or reaction enhancers such as Betaine could be added (16).

An issue we faced in the lab was primer dimerization. There are several ways to avoid primer dimerization including optimizing the reaction through our polymerase and enhancers such that there is an abundance of amplicon for primers to anneal to, adjusting the primer concetration or re-designing new primers with varying GC content to optimize annealing temperatures. We decided to minimize the amount of primers by using a universal set for conserved rRNA regions rather than a set for each pathogen, however this is still an issue that needs to be considered.

Another key feature in producing our device is ensuring that the LAMP reagents can sit at room temperature. A possible solution could be to lyophilize (freeze dry) the reagents using a simplified lyophilization procedure demonstrated by Song et. al (2022). This method involves lyophilizing all of the regents in a one-pot mixture and was shown to preserve the reactivity for over 4 months. Continual testing under uncontrolled conditions such as a wider range of temperatures will be required.

The final concern we have is the color change. We have worked on optimizing the color change of HNB from sky blue to purple through creating more amplicon through our fusion protien, however it can still be argued that the color switch is not distinct enough. To achieve a better colorimetric signal, an interesting alternative would be to explore nanoparticles as they don’t require equipment such as a flourometer and can give off distinct color change.