(A)BME100 was assigned the task of testing 34 patients for a specific disease-associated SNP, and the class was divided into 17 teams of 6 students each so each team was assigned 2 patients to test. One person in the group was in charge of labeling and keeping track of the primers, PCR mixes, and controls, and a second person was in charge of using the pipette to make the PCR reaction. For the second part of the test using a fluorimeter, another person in the group was in charge of pipetting the PCR reaction onto the slide while another group member set up the box, height of the stand, and calibrated the phone for the fluorimeter. The rest of the group members were in charge of taking images of the drops in the fluorimeter and analyzing them using ImageJ. (B) In the first half of the PCR test multiple PCR reactions were tested per patient to increase accuracy, for example a PCR reaction was made for a positive and negative control, then three identical PCR reactions were made for patient 1 and three identical PCR reactions were made for patient 2. The positive and negative controls were made to test the patient PCR results against because the positive control contained the desired SNP while the negative control did not. In the second half of the PCR test, ImageJ was used to analyze the drops of PCR reaction, so certain measurements needed to be kept constant for all of the reactions. These measurements included area, integrated density, and mean gray value. For every PCR sample, three photos were taken of the drop in the fluorimeter and this was done to get the clearest image for use in ImageJ. (C) After running all of the PCR reactions, 30 successful conclusions were made on the diagnoses of the assigned patients. 13 patients tested positive for the disease-associated SNP, 17 tested negative for the disease-associated SNP, 2 tests were inconclusive and 2 groups had no test. Since this was the first time many members of our group had used a micropipette there were issues in the beginning with using it correctly and this could have caused inconsistencies in our data. With the fluorimeter, it was difficult to get the phone to focus on the PCR drop and at the right level because the phone sat at an odd angle in the holder. The box that covered the fluorimeter was also inadequate because it was nearly impossible to take a picture on the phone without allowing light into the box, which affected the images of the PCR drops and hence the data.
What Bayes Statistics Imply about This Diagnostic Approach
Calculation 1 gives the probability of a positive test conclusion given a positive PCR reaction and it is implied that the individual PCR replicates were about 77% accurate for concluding if a person has the disease SNP. Calculation 2 gives the probability of a negative test conclusion given a negative PCR reaction and it is implied that the individual PCR replicates were about 89% accurate for concluding if a person did not have the disease SNP. Together, calculations 1 and 2 imply that the individual PCR replicates are reliable for concluding that a person has the disease SNP.
Calculation 3 gives the probability of a patient developing the disease given a positive test conclusion. The accuracy of PCR for predicting the development of the disease was low, near 20%. Calculation 4 gave the probability of a patient not developing the disease given a negative test conclusion. The accuracy of PCR for predicting that a patient will not develop the disease based on a negative test conclusion was very high at almost 100% accuracy. It is hard to determine the reliability of PCR in general for predicting the development of the disease using both calculation 3 and 4 because the two calculations were drastically different. Calculation 3 shows low reliability in diagnosis while calculation 4 shows very high reliability in diagnosis.
Intro to Computer-Aided Design
TinkerCAD
Our Design
[Image:Micropipette_group15_updated_design_1030.jpg|200px|Micropipette_group15_updated_design_1030.jpg]
Our group re-designed the fluorimeter to use in the PCR lab because the fluoimeter is vital to collecting data that will generate a diagnosis, so a more efficient and easy to use fluorimeter will make this test quicker and more accurate. To modify the fluorimeter, we added a tripod that can hold the phone and designed a box that lets little light in and is easier to use. The tripod holds the phone upright and keeps it stable, so that it is easy to align the phone at a ninety degree angle to the drop. With the added stability of the tripod, the phone will not move while photos of the drops are being taken so the photos will be clear and more accurate. Since the SYBR green in the drops requires darkness to work with the flash of the camera to appear, our group came up with a box design to keep it dark. The box will cover the apparatus with the drop as well as the tripod with the phone, and feature a sliding window so that the photos can be taken in the dark. The phone's camera can be set up with a timer once it is in the tripod and underneath the box, then the window can be shut while the pictures are being taken.
Feature 1: Consumables
PCR reaction kits include "very important" consumables which are non-machine materials that are necessary for a PCR reaction and for testing for disease-associated SNP's. These consumables include PCR mix, primer solution, SYBR green solution, buffer, patient and control samples, glass slides, plastic tubes, and pipette tips. Our company's PCR reaction kit will include all of the items listed above, but two of the plastic tubes will be totally black instead of clear because these two tubes will be for the SYBR green solution. These black tubes will solve the issue of the SYBR green solution being exposed to light, because the solution is sensitive to light. This product will go along with our redesigned box used with the fluorimeter which also keeps the SYBR green solution out of the light so that it shows up better in images.
Feature 2: Hardware - PCR Machine & Fluorimeter
Our company will include both the Open PCR machine and the fluorimeter in our system, however the fluorimeter will be redesigned because the original design is inaccurate and difficult to use. The open PCR machine will be used just as it was in the lab because we found this process easy to follow, and accurate results were obtained as well. A fluorimeter with a tripod to hold the phone, and a design to keep light out will also be included in our system. The fluorimeter needed to be redesigned because too much light was getting into the box and affecting the clarity and accuracy of the photos of the samples.
BME 100 Fall 2015
