Team:Worldshaper-Shanghai/Implementation
Proposed Implementation
Introduction
Through our research and meaningful work during Human Practice , we discovered that the early detection of any abnormal signs or data during physical examination has been the most effective way on both diagnosis and treatment of prostate cancer. Apart from our interviews with the experts, the team launches investigations on public opinion through online surveys as well as face to face interviews. The results turn out that a majority of the population demonstrated a surprisingly high rate of psychological repellence and distrust against the current ways of prostate cancer diagnosis. Therefore, we decided to create a new way of time-efficient, inexpensive and non-invasive method of prostate cancer diagnosis to replace the existing detection methods such as DRE (digital rectal examination), PSA (prostate specific antigens).
Based on our research, major hospitals in China are arranged in a way that the systems only focus on the efficiency of the hospital rather than patients, so the time consumption of actual conference with doctors weighs much less than the time consumption of registration and waiting in line. Therefore, our projects are designed to be put in to Community hospitals or physical examination centers that are closer to patients. In such a way, patients will be able to minimize their time consumptions during diagnosis while also remaining efficiency of hospitals by transferring some projects to community health centers. The trained personnels of clinical laboratory here can not only ensure the normal use of our products but also avoid cross contamination caused by use of ordinary people.
Product guiding
1. Preparation before use
Users need to prepare the following equipment and reagent: different sizes of pipettes, a small centrifuge, a metal heater, and absolute ethyl alcohol.
2. Consumable Materials in the kit
① 10ml Urine Collection Tubes (×20)
② 1.5ml Tubes: For Tube B mentioned in the process.
③ Disposable Gloves
④ Hand-sanitizer
⑤ Magnetic separator
3. Reagents in the kit
⑥ DEPC H2O 5 ml
⑦ Tube A (×20): 10ml tube with 5 ml of a mixture of magnetic beads and absolute ethyl alcohol
⑧ Tube C (×20): Tube C has a mixture of RT Buffer, Rnase Free H2O, Oligo dT Primer, Random 6 primer and RT enzyme Mix（RT reaction is based on PrimeScript™ 1st Strand cDNA Synthesis Kit from TAKARA）
⑨ Tube D-PCA3 (×20): Tube D has a mixture of RPA Mix（Rehydration Buffer, MgAc）, PCA3 Primers and ddH2O (RPA reaction is based on TwistAmp Basic Kit from TwistDX)
⑩ Tube D-KLK3 (×20): Tube D has a mixture of RPA Mix（Rehydration Buffer, MgAc）, KLK3 Primers and ddH2O (RPA reaction is based on TwistAmp Basic Kit from TwistDX)
⑪ Tube E (×40): Tube E has a mixture of Toehold DNA, S30 premix Plus, S30 extract and DEPC H2O (S30 T7 High-Yield Protein Expression System from Promega)
⑫ Positive control (×20)
⑬ Negative control (×20)
4. Standard Protocol
1) RNA extraction ：
Reagents we need:
·Tube A-Magnetic Beads
·70% ethanol
·DEPC H2O
① Collect more than 5 ml of urine with the given tools.
② Add 5 ml of urine into Tube A, then lightly shakes the test tube upside down for a few time (about five times).
③ Wait for 10 min at room temperature.
④ Move 1.5 ml mixture from Tube A into a new clean centrifugal tube (Tube B). Then put Tube B on the magnetic separator been given. Wait till you see the magnetic beads accumulated on the tube wall and remove all supernatants.
(Repeat step 4 until all mixture in Tube A is moved)
⑤ Add 70% ethanol into Tube B, wait for five minutes at room temperature, and the tube shall not be placed on the magnetic separator while waiting for this step.
⑥ Put the Tube B on the magnetic separator again. Wait till you see the magnetic beads accumulated on the tube wall. Then remove all supernatant.
⑦ Transient centrifuge the Tube B for about 30 s and remove all liquid from Tube B.
⑧ Place the Tube B at room temperature for 5 min to dry the magnetic beads. M
⑨ Add DEPC H2O to Tube B, tab the bottom of the tube lightly to suspend magnetic beads. Dissolving RNA for 5 min.
2) RT:
Reagents we need:
·Tube C-RT reagent
（Positive control and Negative control can be used since this step）
⑩ Move the RNA from Tube B to Tube C, wait for few seconds to let the supernatant mix with solutions in Tube C.
⑪ Put the mixture in Tube C onto the heater, turns the heater to 42 ℃ and wait for 1h to let the RT reaction finish.
3) RPA:
Reagents we need:
·Tube D-PCA3
·Tube D-KLK3
(RT product needs to react with Tube D-PCA3 and Tube D-KLK3 respectively. Take Tube D-PCA3 as an example)
⑫ Extract RT product from tube C into the Tube D-PCA3, and then wait for a few seconds to let the solutions mix well.
⑬ Then put tube D-PCA3 on the heater again with 37 ℃ for 15 min to let the RPA process finish.
4) In vitro tanscription and translation
Reagents we need:
Tube E-Vitro tanscription and translation
⑭ Move the RPA product of PCA3/KLK3 from Tube D into Tube E. Wait for few seconds to let the solutions mix in Tube E.
⑮ Put Tube E back on the heater again for 3h with 37 ℃.
⑯ see the resulting color of the product：
Red the color of the tube after heating
a)Red indicates a potential risk for prostate cancer
b)No color change indicates risk-free.
Worldshaper-Shanghai 2020
New non-invasive technique for early stage prostate cancer diagnosis