Design
Since the aim of our project was to create an easy-to-use on-site test for BRD, we didn’t want to use traditional DNA amplification methods like PCR. Conventional PCR requires expensive and relatively large thermocyclers. Isothermal methods, like LAMP or RPA, don’t require this equipment.
We chose to use RPA or recombinase polymerase amplification as our detection method. This method is easier to use than other isothermal techniques, since it only requires two primers (Daher et al., 2016). Simply, RPA components are combined with target DNA. Recombinase proteins bind to the primers and help to scan the DNA for complementary sequences (Tan et al., 2022). Primers anneal to the target DNA and single-stranded DNA binding proteins then stabilize the displaced DNA strand, preventing the ejection of the annealed primer. DNA polymerase can then extend the primer using the target DNA as a template. No temperature changes are required and the whole process can take place at temperatures ranging from 20 to 42°C. RPA is an affordable, simple, and fast method for DNA detection (Sumit Jangra et al., 2021; Magrina Lobato & O'Sullivan, 2018).
For our first proof-of-concept experiments, we utilized the TwistDx (Cambridge, UK) TwistAmp Liquid Basic kit. This kit provides all the RPA components in easy to use liquid form and costs $300 CAD for 100 reactions.
The amplified DNA from RPA reactions are usually detected using agarose gel electrophoresis. However, we wanted to investigate the use of hydroxynaphthol blue (HNB) as a colourimetric detection method. This chemical is a metal-sensing dye that changes colour in the presence (or absence) of magnesium ions. During DNA amplification, DNA synthesis generates inorganic pyrophosphate (PPi) as a byproduct. PPi in turns binds to Mg2+ ions and precipitates out of solution as Mg2P2O7. As magnesium is pulled out of solution, it can no longer interact with HNB and the colour changes from violet to sky blue. However, it is important that the correct form of HNB is used. Both the disodium and trisodium salts are available for purchase, but it is only the trisodium version that can under the correct colour change.
In practice, Bo-Find would require vets or farmers to take a deep nasopharyngeal swab of the cattle. DNA from the collected samples is traditionally isolated using a commercial kit, such as DNeasy Blood & Tissue Kit or QIAmp PowerFecal DNA Kit, both from Qiagen. However, we proposed that may be unnecessary and a simple heat treatment step could be used instead. The swab would be placed in a microfuge tube with a PBS buffer. The tube would then be heated to 100°C for 10 minutes. A defined volume would then be transferred to the prepared detection tubes. Each detection tube would contain the RPA components and a different set of primers for pathogen detection or control reactions. Our Bo-Find device would then incubate the tubes at 37°C for 30 minutes and a colour change due to DNA synthesis would occur in any positive samples.
While superior in theory, the kit’s design will need to be tested and improved for practical use. The device’s current design allows for testing of only one cow at a time. This can make Bo-Find unfeasibly slow if large-scale detection is required, so we plan to find a way to improve the design to allow for faster testing of multiple animals at once. To overcome these potential problems we can consider developing a larger testing device that will be able to hold more rows on test, thus allowing for less waste while still testing the same amount of cattle. In feedlots cattle are usually divided into multiple pens. In these pens they are in close proximity to each other. Because of this, they develop very similar microbiomes within around 14 days from initial arrival at the feedlot. Consequently, only one to three cows would need to be tested per pen, depending on the size of the pen. This would allow for use of less tests, creating less product waste and less time for farmers or researchers to test each cow.
