Overview of the Original Diagnosis System
In the conducted experiment, PCR was used in order to test whether or not a specific DNA sample contains a disease-specific SNPs. There were 10 groups in total testing 20 samples of DNA for the SNP. Each group tested two samples each with three trials per patient. In obtaining the results, there were several methods set in place in order to prevent errors. For example, each DNA sample was replicated three separate times and the results were tested. This helps to reduce the chances of outliers in the data since there is an average of three that can be taken from the data. The positive and negative controls were also set in place in order to ensure that the PCR machine is working correctly as well as set a relative amount to compare the other data to. The ImageJ values in the lab were also calibrated and three trials (or pictures) were used to account for variation. Within this lab, it was possible that since each of the pictures taken were not all at the exact same angle, that it is possible that there is some error in the PCR concentrations calculated.
What Bayes Statistics Imply about This Diagnostic Approach
The results for calculations 1 and 2 were close to 1.00 (100%) which implied that the reliability of the individual PCR replicates for concluding that a person has the disease SNP or not was relatively high. However, the reliability would increase exponentially with the number of patients that are tested; with more test results, the percentage of reliability increases proportionally. The Bayes values could be more reliable with an increased amount of patients, however, not completely reliable in diagnosing the patient due to potential fluorescence errors in the lab.
Calculation 3’s results showed that close to 50% of people that obtain a positive test result will actually have the disease. This implies that the PCR system is not reliable at determining if a subject has a targeted disease/DNA identifier. However, calculation 4’s results implied that the PCR system is reliable at determining whether a subject does not have a targeted disease/DNA identifier. The calculation showed that close to 90% of subjects that are given a negative test result will not actually have the disease.
Three possible sources of human or machine/device error that could have occurred during the PCR & detection steps that could have affected the Bayes values in a negative way included the use of different phones throughout each significant group. A number of the groups obtained their experimental results with phones that had better picture-taking abilities, and therefore their values may have been comparatively more accurate or reliable. Also, human errors could have been significant during the picture-taking process or the micropippetting portion of the experiment. Moreover, the fluorescence of the drop may have been reduced in the picture because the system was not completely submerged in the dark; therefore, the light in the room may have affected the results calculated from the pictures in ImageJ software.
Intro to Computer-Aided Design
3D Modeling
The software that was used to replicate the new idea that was constructed based on the original PCR machine was Solidworks. Since the idea was completely different from the original idea, Solidworks was used to be able to create the intricate designs that were needed. Solidworks was a great way to incorporate ideas into in a 3D image without having to build the object itself. The more practice an individual has with Solidworks the better the experience can be. Although it is more of a professional software, it is much preferred because it allows for more control of the creation.
Our Design
The image above is the design of a rapid-test fluorimeter device that would process a sample of DNA from a patient and test for a single nucleotide polymorphism or disease. The device would come with a care package that contains cuvette and a top where the patient's DNA is placed into the cuvette than inserted into the top compartment and covered with a provided cap.This design allows for an easier method to diagnose a patient in a rapid and hassle-free way. The device is much more compact, about 4 by 3 inches, and would be able to instantly give results in a matter of minutes compared to an hour to two hours. The device would connect to a computer with a software that would process the information that was inputted into the device and give comparative results of the patient's DNA to a non-disease DNA sequence. There would be no picture-taking process required, no hassle of trying to get a focused picture, and no extra work needed to try to understand the fluorescence of a picture that isn't always accurate due to the variability of the phone. The device would examine the patient's DNA using an algorithm that would locate the SNP and diagnose the patient.
The device is a modified fluorimeter that connects to the computer through a USB. This design allows for the PCR product to be loaded through a cuvette into the device and is analyzed by a measurement of how much light passes through the sample. This is different than the smartphone method because it does not need ImageJ in order to analyze the samples. We chose this because it is more accurate and faster than the smartphone method of analyzing PCR samples.
Feature 1: Consumables
The consumables kit will be comprised of 3 single boxes that will contain most of the materials needed for a PCR lab. The first box will contain a single micropipettor so that it is easy to keep it in one place. The next box will contain 200 individual micropipette tips in an organized 20x10 formation. The third box will consist of 6 rows and 10 columns of the other materials, including cuvettes, test tubes, PCR mix, the first primer, the second primer, and the DNA template samples. The top row of the bottom box will hold the customer-supplied DNA template samples that will be used for the PCR lab. The next row will hold one primer and the following row will hold the other primer. The next row below will contain the PCR mix. The last row will contain the cuvettes for the fluorimeter lab and each cuvette will have a blank space available for labeling.
This design addresses the problem of how it is easy to get lost in the multiple steps of preparing the DNA samples for PCR. In this design, the content in each row is moved to the test tube below in order from top to bottom. Once all 4 rows of prepped material are moved into the test tube, the test tube can be put in the PCR machine and the content then transferred to the cuvette in the bottom row of the sample’s column. It is therefore simple to prep and transfer materials.
Feature 2: Hardware - PCR Machine & Fluorimeter
As for the actual PCR machine, our group has chosen not to alter the design since the actual machine has few weaknesses. The machine simply needs to fluctuate between set temperatures for certain amounts of time and spending more money for a nicer machine that may have slightly more precise temperatures was determined to be not worth it. The fluorimeter however has several weaknesses considering the accuracy of the results obtained, so our group has chosen to change the design. In our design, the fluorimeter is altered to be more precise in measurements of the concentration of DNA in the PCR results. The device will be handheld and will plug into the USB port of the computer. A cuvette filled with the PCR product will be inserted into the device and it will be activated to measure the concentration of DNA in the sample and the data will be sent to the computer. Before sending information to the computer, it will calculate the concentration of the DNA based on the amount of light that passes through the solution.
In the current design, the results are easily distorted based on the location of the smartphone since the angle of the picture was changed in between the photos, changing the results. Additionally, ImageJ is a very tedious process with a large possibility for error in the analyzation process. Because of the tedious calculations and the inconsistency of the samples, our device eliminates these issues by first eliminating the smart phone camera discrepancies and analyzing each sample in the same location each time and under the same conditions. The device also aanalyzes the DNA concentration much faster and more accurately than the ImageJ program since it eliminates the possibility of human error.
BME 100 Spring 2018
