The analysis was done on 26 teams, each of which had two patients to sample. This leads to a total population size of 52 patients. PCR of course was essential, without it no analysis would have been possible. The samples from the patient were placed into a PCR reaction with a selective primer that will only bond and replicate DNA that matches the mutation being searched for. Then, sybyr green dye was added to the mixture and photographed in a dark room, giving us a consistent way to measure DNA presence. Furthermore, each patient had 3 samples taken, allowing for extensive cross checking and preventing any singular contamination from slipping past without throwing up a red flag. Furthermore, the ImageJ Software was extensively calibrated. Using multiple samples of known DNA concentrations we established an equation that related the amount of green light coming from a sample with the concentration of DNA that it implied was present. Thankfully, all samples between patients returned similar results, indicating there was not a cross contamination.
What Bayes Statistics Imply about This Diagnostic Approach
Of course, any diagnostics tool is only as good as the results it produces. Furthermore, no test is perfect. However, this test is very successful. Bayes values were close to 1.00. Furthermore, a testing method is always susceptible to human error. This test manages to avoid a lot of that by using simple, easy to replicate methods to prevent error. With a simple rule of labeling all test tubes and using a new pipette tip, human errors can be effectively eliminated. Other than a malfunctioning PCR machine, there are few ways for this test to completely fail, and with positive and negative tests in each batch and 3 samples per patient, these risks are further and massively reduced.
Likewise, diagnostic probabilities were good. Odds were high that given a positive test the disease would develop and that given a negative test the disease would not. As an initial screening method, this very effectively focuses attention on those most likely to develop the disease.
Computer-Aided Design
TinkerCAD
The TinkerCad tool allows people to design and create three dimensional objects. The interface is very user-friendly, which helps with the creation of simple or even complex objects. This tool is especially helpful when it comes to 3D printing. For the lab, TinkerCad was used to create part of the open PCR device. The entire PCR machine could be created, but only the front, back, bottom, and one side were required.
Our Design
Open PCR Machine PC Easy
Inside view of Fluorimeter PC Easy
Fluorimeter image with cover PC Easy
Our design of a new PCR machine and new fluorimeter improves some of the identified weaknesses of the existing PCR machine and fluorimeter. First, with the new PCR machine, it will be able to run 24 samples rather than 16. This will help reduce some of the time needed to analyze many samples. Yet, the machine will increase in size only slightly, in order to keep it compact. All of this helps the new PCR machine remain cost-effective, since more samples can be analyzed with just one machine and the cost of making it will not jump too high. Second, our new fluorimeter device comes with new features as well. One of its key features is the fact that the user can load three slides with three samples on each slide into the device. Each chamber of the device is isolated from the other, so that no light from other chambers influences the sample's image. Another key feature of our device is the integrated sliding camera. The camera will be able to take pictures of each chamber when the user manually opens the lid and slides the camera into position.
Feature 1: Consumables Kit
The packaged consumables that will be included in our kit are:
PCR reagent mixers
test tubes for samples
24 test tubes that can hold 50 µL of mixed Taq DNA Polymerase, MgCl2, and dNTP's
24 empty PCR tubes
24 tubes of 50 µL DNA/primer mix
SYBR Green I Dye
Multiple pipette tips (100)
Micropipettor
Glass Slides for Fluorimeter (50)
The consumables will be packaged in an organized fashion in order to ensure each customer receives the right amount of materials and so that the customer has an easier time managing those materials. The consumables will be separated into separate sections, with some of them in the same section so that we can maximize the space within the box they will be placed in. The micropipettor and pipette tips will be in the same section. The test tubes will be placed in the same section. Everything else that is left such as the glass slides will be placed in their own section. There are also enough consumables to fully run 24 PCR reactions, which is the full capability of our devices. Lastly, the box will come with a label that states the quantity and type of items that are included in our consumables package.
The major weakness with the consumable packaging plan is the fact that so much of the plastic is thrown away during experimentation. Our consumable package plan will try to reduce this by using more environmentally sound materials in the production of the consumables. Additionally, our group plans on offering the choice of ordering less tips or empty tubes if the customer already has these materials and does not require them. Another option we have considered is making the tubes and tips so that they are reusable through sanitation. Additionally, if customers wish, they will have the option of sending us their used tubes and tips, which we can sanitize and re-sell to customers who wish to buy them. This can help our company become more sustainable since our resources may not always have to be manufactured 100% of the time. Our company will also gain an eco-friendly image from this service.
Feature 2: Hardware - PCR Machine & Fluorimeter
The PCR machine and fluorimeter will be included as separate systems that are key to our system. The PCR machine is used to replicate DNA while the fluorimeter is used to analyze that replicated DNA. This process is key when looking at DNA for diseases. The PCR machine must be connected to a computer also, which does not present too much of a problem, especially if working in a lab. Our PCR machine and fluorimeter will be packaged in separate sections within a box, so that their contents can remain isolated yet secure.
The current PCR machine only allows for 16 samples to be replicated during use. This presents a huge problem when multiple samples need to be replicated. The PCR machine that we have created will be able to replicate 24 samples. Additionally, the size of our device will not increase dramatically so that it does not become too bulky for customers to move if needed. Our PCR machine will also pre-set parameters so that customers can choose to use them if they like. Otherwise, they will still be able to change those parameters to their desired settings for their own replications.
The current fluorimeter only allows for one slide to be placed down with one sample. This can be time-consuming, especially, when multiple photos need to be taken of the sample or if multiple samples must be analyzed. Our new fluorimeters has the capacity for three slides with three samples to be analyzed quickly and effectively. Additionally, our fluorimeter contains an integrated camera, so that customers do not have to worry about using a smart phone camera at the right angle and view of the sample. The integrated camera will be able to slide down a track so that it can take multiple pictures of each chamber that has its own sample. Another advantage with this integrated camera is the fact that the customer will not have to worry about setting a timer and blocking the light from getting in to the box. With the design of the lid of our fluorimeter, no light will be able to get in to the box.
BME 100 Spring 2015
