When initially designing our project, we decided it was best to stick as closely to the current methods for testing bTB as possible. We decided this due to the fact that the stakeholders involved are reluctant to change and have been using their current systems for many years. However as the project went along, and after speaking to the stakeholders involved, it became clear that they would be receptive to change. This allowed us to design and construct both a new test and testing method. These have been heavily influenced throughout the project by the stakeholders and are discussed below.
Design 1 - the Eppendorf
We first assumed that our testing could take place in a 1.5ml eppendorf tube. Our cell free system currently takes up a volume of 50μl, leaving ample space for a few drops of blood to be added. If RNA in this blood sample is associated with bTB, it would trigger our toehold switch, therefore causing a reporter to be expressed (eg, a colour change). An eppendorf tube is lightweight, durable, and can be sealed to prevent outside contamination. This seal is an important design feature, as the environment in which the test is performed can be unclean and full of possible contaminants. This was also chosen as we thought we could extract blood from a cow using a skin prick test, giving just a few drops of blood. However on consultation with Phil Leighton, he told us this would not be possible due to a cow's thick hide. This led on to our second design, which allows for a standard blood test to be used.
Design 2 - the grip tube
Following a meeting with Phil Leighton, a vet who conducts bTB tests in South West England, we realised our prototype needed some major changes. We learned that blood tests in cattle take place behind the tail, as opposed to the current skin test which takes place in the neck. According to Phil, the blood test is easier and safer to perform, however there is still a partial risk of kicking from the cows hind legs. This risk has been significantly reduced as the cow is normally held in a crush, preventing the rear legs from kicking backwards and letting the tester stand to the side of the animal. However, he also told us that an eppendorf tube would be too small and fiddly for the purpose of a blood test. The current blood test tube is around 110mm long with a 12mm diameter, bulky enough for the vet performing the test to get a good grip on the tube whilst drawing blood from behind the cow. The environment in which the test takes place is unclean and hectic, so our next design took elements from the current blood test tube. We designed a larger test tube with a vacuum sealed lid, shown on the left. Inside the main tube is a smaller eppendorf tube containing the cell-free test system.
Design 3 - the filter
Our own research into bovine blood testing and further advice from Phil brought up a new issue: bovine blood tests can draw up to 7ml of blood. Our test only requires a few drops, but the vet performing the test would not have time to measure delicate amounts of blood whilst behind an agitated cow. So we decided to move the eppendorf containing our cell free kit to the base of the blood tube. We then needed to design a filter that allows only a small amount of blood to interact with our test. Ideally this membrane would let only a few drops of blood through whilst keeping the remaining blood above the membrane. This method lets the vet collect a blood sample as normal. A further benefit is that in a future design the cell free kit could snap off of the blood tube, therefore allowing both a TB test to be carried out and a blood test. By associating these two parts together, most likely with a tear off label, the blood could be tested in more detail if required.
The 3D mockup shows the Vacutainer with an eppendorf in the base containing our cell free kit with the filter situated above this. This design allows the blood to be kept away from the cell free kit whilst the filter lets the correct about of blood into the eppendorf tube. Hopefully, this should allow the vet to take a blood sample as normal, but also ensure the cell free kit isn’t saturated with blood.
Design 4 - More Suggestions
The membrane in design 3 was selected such that it would allow a certain volume of blood to flow through it and into the cell free system. However after consulting with various advisors we realised this would either be very expensive or not even possible.
Upon speaking to Prof Peter Winlove, professor of Physics at our university, although initially impressed with the idea he again reinforced the above opinion. However he proposed a solution to the problem, by changing the membrane to a pore then using hydrostatic pressure it should also only let a certain volume through.
Below is the maths used to calculate the necessary parameters to make sure that the required amount of blood (ΔV) flows through into the cell free kit.
The top of a Vacutainer
showing the material
proposed for use in the
ViPi = VfPf
Vf = Vi - ΔV
Pf = Pi + ρgh
(Pi + ρgh)(Vi - ΔV) = PiVi
He also mentioned that we would need to have an anticoagulant to prevent the blood from clotting during the test. He informed us that we could purchase tubes with this already in them, hence simplifying the whole process as we could just purchase these.
Another thought proposed by our very own team member Joe was to use a membrane based system as described above. However we would design it so it was made of a similar material to lid of the Vacutainer. Then by training the vet to insert the needle through both the lid and the membrane letting only a few drops though first in the cell free kit, pulling the needle back into the top chamber to release the remainder of the blood. This prevents saturation of the cell free kit and provides a low tech, low cost solution to the problem form design 3.
The Current Methods and Their Limitations
The final prototype of our bTB test has been honed and revised several times as we considered the practicalities of TB testing for the vet performing the test. We took in to account advice from vets local to South West England (namely Phil Leighton and Dick Sibley) and incorporated this into our prototype design. This allowed the design to ‘evolve’ and advance; we now have a final prototype which should maximise the ease of testing and minimise any error in the testing.
Firstly we had to consider where we would take the testing sample from. The current skin test is performed on the neck, which must be shaved and then injected with avian and bTB. The vet then returns 72 hours later to diagnose the cattle based on the skin’s reaction to the injected strains. Phil told us that neck testing is awkward and time-consuming for the vet, and that blood tests are easier to perform.
As the RNA associated with a TB infection (but not present in the BCG vaccine) is found in the blood of infected cows, we further discussed this option with Phil. He told us that bovine blood tests are usually drawn from a prominent vein behind the tail of the cow, and that this kind of test has many benefits over the cumbersome skin test. These are outlined:
- The blood test is much easier and quicker to perform. There is no need to shave the area, it is a simple process of inserting a needle and drawing blood.
- The blood test is a safer option, as the vet can stand to the side of the cow (avoiding kicks from the hind legs) to draw blood quickly, and the thick skin in this area means that the cow is unlikely to feel much pain from the test.
- The current skin test can agitate the cow as the neck is a sensitive area, which must be shaved and injected.
- The vet must only make one visit to the farm to draw blood samples, whereas with the current test the vet visit twice returning 72 hours after injection to diagnose.
We then focused on making our test as easy as possible for the vet to perform. Phil Leighton was invaluable during this process, telling us exactly how we could help vets like him. The current test has several steps: the cow arrives through a press, the number on the cow's ear tag is recorded, it is shaven and injected with the attenuated strains, and then released. 72 hours later, the vet returns. The cow again arrives through a press (in a random order), it’s ear tag number is recorded, the neck is observed and then finally the cow is diagnosed and the appropriate action can be taken. This method has several areas where error can occur:
- The wrong number could be written down at either stage (testing or diagnosis).
- The list of numbers written down can be mixed up, resulting in the diagnosis being matched to the wrong cow.
We aim to help these problems by devising both a new test and a new method of testing. The testing methods contains both a prototype for the test and a method for the test.
Our new test
Our test has fewer steps and should be fairly quick and easy in comparison with the current test. First, the cow arrives through a press, it’s blood is quickly drawn into the test tube, the tube is then labelled with the cow’s ear tag number, and the cow is released. The risk associated with getting the blood is also reduced as the crush prevents the cow from kicking backwards. It also lets the vet stand to the side of the animal when gathering the sample. After a few hours, a diagnosis can be made from the labelled test tubes. As a result of this, the only possible error source is from incorrect labelling or contamination.
Once the RNA associated with a bTB infection has been detected, a reporter gene is expressed. We wanted this reporter to be immediately clear and visible to any untrained personnel eg. fluorescence was unsuitable as the equipment required to visualise fluorescent proteins is expensive, requires training to use, and is not portable.
Firstly we decided on a simple colour change using chromoproteins. However, taking into consideration the nature of the sample - blood - we were unsure as to whether a colour change would show through the dark colour of the blood. This could be solved by centrifuging the blood and testing the plasma, which has much less colour, so would interfere with the chromoproteins to a much lesser extent. However, we were adamant that our test should require no technical knowledge or equipment, so we have came up with several other possible solutions which we are unable to test due to time constraints:
- Using a metal strip which the chromoprotein could aggregate around, causing a colour change to the strip which could then be removed and examined.
- Using a paper-based system, where a drop of blood could be placed onto a cell-free piece of paper and a diagnosis could be made.
- Using an enzyme indicator - eg. an agglutination assay.
Our New Method
Not only is the current test for TB outdated, but so are current methods of testing. Currently the cows are recorded using ear tags, these are either plastic or metal, and attached to the cows ears. There are normally two of them and the numbers on them must be written down once the cow is tested. However this is a very analogue way of recording the information.
The method we propose is to change the ear tags so that they contain a passive RFID tag, as well as a number. This would allow a lot more information to be contained on the tag. In addition, it also changes the system to a digital version whilst still keeping it familiar to the farmers that use it day to day.
By placing an active RFID reader in the testing facility, this could be used to power the passive RFID tags located on the ear tags. The reader can work up to 100m, whereas the tags work up to 10s of centimetres. If this was placed at the crush containing the cows then as they went through they would be registered as being tested. This removes the chance of human error and also speeds up the testing procedure. The number on the tag would still have to be written on the test tube to associate this with the animal tested.
We were reluctant to have much human involvement, but after much deliberation is was decided that writing on the tubes would be the easiest way to link the cow and the tube together. The RFID tag would greatly speed up the cataloguing of testing.
After waiting for our test to be finished, the positive reactors could then be located and the number found. By inputting these numbers into the computer it would be possible to then walk the cows back through the crush and have an automatic signal for when the infected animal passes through. So therefore it would be simple to find it and remove it from the herd.
For an existing farm passive RFID tags would have to be purchased for the herd and one active RFID reader located at the crush. A free piece of software would then be downloaded onto a laptop to log the cows and locate the infected animals.
The cost for these devices are as follows:
- Passive RFID $0.15 each, which are stickers so can be used on existing tags 
- Active RFID beacon can be purchased for a maximum of $100 
bTB costs the UK economy £1 billion every year, and this cost is steadily increasing. Two significant parts of this sum are the costs of testing cattle, and those of compensating farmers.
A huge part of the costs of bTB is the compensation paid to farmers. When a cow tests positive for bTB, further testing is carried out, and then it is slaughtered if still found to have bTB. The UK government compensates the farmer for any cattle slaughtered. Although this sum is often much less than the actual value of the cattle, it costs the UK a huge amount every year. In 2009 total compensation costs were over £50 million. The carcasses of slaughtered cattle are checked for bTB, and any that do not have tuberculosis lesions in more than one organ or body part are sold into the food industry. This helps to reduce the compensation costs by a small amount.
DEFRA employs vets to perform regular tuberculin skin tests on UK cattle. This occurs every four years in low risk areas, but can be higher in areas with a major bTB problem. If a herd is found to contain any animals which react positively to the skin test, restrictions are imposed upon the herd and another test is performed in 60 days. This testing continues every 60 days until the herd is proved to be clear in two separate tests. The UK government provides information on the average amount testing costs every year. The current test costs approximately £5.90 per animal in 2001/10 .
Cell Free Kit:
The cell free solution (S30):
- 30 reactions for £342 
- Per reaction - £11.4
- 250 reactions for £199.82 
- Per reaction - £0.80
Initial toehold order (S30):
This is the cost to order your custom designed toehold which only needs to be purchased once, as it can then be remade from the plasmid. The price used is the price of our own toehold which was ordered through IDT.
- Our toehold £140 
Toehold running costs:
These are the day to day lab costs incurred when producing the toeholds from the ordered plasmid. If this was produced in an established working lab, these cost would be minimal as the consumables used to make our test are used every day.
- Agar Plates
Total Cost for Cell Free kit:
Per reaction - £12.2. This is in addition to the setup cost of £140
BD Vacutainer Plastic Blood Collection tubes:
- 100 tubes for $34.87
- Per tube - $0.35 (£0.23)
Microcentrifuge tube Safe-Lock 1.5mL:
- 1000 tubes for £40.15 
- Per tube - £0.04
Using design 4 we thought we could get this custom molded to our specification. This means that after we've constructed a mould for this shape, our production volume would be able to keep the cost of this down. As we are unable to find an exact cost for this, an estimate based on the cost of the current tube can be used. We thought we would double the price as it would be a relatively new container.
A detailed analysis of the cost break down of iGEM-Bielefeld-CeBiTec teams custom made cell free kit .
- Per Container - $0.70 (£0.45)
Total Cost for The Container:
Per container - £0.72
Cell free kit per reaction - £12.2
Container per container - £0.72
Total cost per reaction - £12.94
Our cost is currently £12.94 and compared with the £5.90 for the current test it is over double the price. Analysing the cost, you can see that the cell free kit is the most expensive part of the test. To try and fix this we reached out to Bielefeld and due to our previous Skype calls with them we knew they were making their own cell free kit. They later provided us with the cost break down of this, which is attached above. As you can see $0.50 (£0.32) is the cost per reaction for one of their cell free kits. If we extended our collaboration with Bielefeld, they may be able to provide us with this cell free kit and hence significantly reduce our test cost.
Using Bielefeld's cell free kit, our overall cost is reduced by £11.08, leaving a new total of £1.84. This easily beats the current testing cost, meaning that our test can not only perform better than the current test, but it is also more cost effective.
 Fischer Scientific, BD Vacutainer™ Plastic Blood Collection Tubes with K2EDTA: Tube Stopper: Tubes. [Online]. [Accessed 15 September 2015]. Available from: https://www.fishersci.com/shop/products/bd-vacutainer-plastic-blood-collection-tubes-k-sub-2-sub-edta-tube-stopper-4/p-179488
 Fisher Scientific, Microcentrifuge tube Safe-Lock 1.5mL write-on assorted from Fischer Scientific UK[Online] [Accessed 15th September 2015]. Available from: http://www.fisher.co.uk/1/1/82080-microcentrifuge-tube-safe-lock-1-5ml-write-assorted.html
 Promega, E. coli S30 Extract System for Circular DNA. [Online] [Accessed 15th September 2015]. Available from: https://www.promega.co.uk/products/protein-expression/prokaryotic-cell-free-protein-expression/e_-coli-s30-extract-system-for-circular-dna/
 Thermo Fisher Scientific, GeneJET Plasmid Miniprep Kit - Thermo Fischer Scientific. [Online] [Accessed 15th September 2015]. Available from: https://www.thermofisher.com/order/catalog/product/K0502
 IDT, 2015. Email to Exeter iGEM 2015, 16th July.
 iGEM-Bielefeld-CeBiTec, 2015. Email to Exeter iGEM 2015, 14th September.
 Jovix (Atlas RFID), Atlas RFID Solutions Active vs. Passive RFID. [Online] [Accessed 15th September 2015]. Available from: http://atlasrfid.com/jovix-education/auto-id-basics/active-rfid-vs-passive-rfid/
 Bovinetv.info, (2012, November 26th). Bovine TB in the UK, England, Ireland, Wales and New Zealand. [Online] [Accessed 16th September 2015]. Available from: http://www.bovinetb.info/index.php
 TB Free England, Learn all about bovine TB in cattle and badgers | TB Free England - TB Free England. [Online] [Accessed 16th September 2015]. Available from: http://www.tbfreeengland.co.uk/home/
 Saved and Safe Ltd., Saved and Safe Ltd. - UK. [Online] [Accessed 20th August 2015]. Available from: http://savedandsafe.org/en/uk