NUDT_CHINA’s Human Practice Tour

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MAP

(click figures to view details of our HP)

This year we expanded our project to Human Practice in three places: Hospital&Health Center, Market and the Public. In each expansion we held meaningful activities closely related to our topic, and this turned out to be a nice interaction between the project and the practice.

Arrows in the map show our practice work in two directions.

Forward Arrows:What the project contributed to the world---a new medical application for Hospitals&Health Centers, a potential handy, low-cost early cancer detection method in the market, and cancer knowledge spreading&surveys and publicity conducted to the public.

Backward Arrows:What we obtained from the world---we did consultation, sample collection and surveys in medical organizations, made a cost evaluation and market demands analysis in the market and got valuable feedback and suggestions from the public on the project.

As a conclusion, we expanded the project to the practice to better serve the world with our creation, and at the same time search for suggestions from the world that could enhance our project. With the interactions between our project and the world, we felt the iGEM work this year was a big issue that influenced not only us, team NUDT_CHINA, but also people around us.

SCHEDULE

:) Click boxes above to see our project & the practice.

This is a specific guide of our practice work this year, listed with the time they were conducted. With our project starting and ending, the HP went on.

PART I

COLLABORATIONS WITH HOSPITAL & HEALTH CENTER

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Considering the fact that our project this year had a close relationship with cancer, and the aim of our project was to create a novel clinical application---handy, low-cost blood-microRNA detetion system, we attached great importance to medical professionalism. Thus we turned to doctors from First Xiangya Hospital affiliated to CSU, Second Xiangya Hospital affiliated to CSU and Health Center of NUDT as our medical professional consultant in the whole process of the project.

Interviews with Doctors

1.Dr.Li Wenjin, Hematology Department, First Xiangya Affiliated Hospital of CSU

(1)Procedure of Current Cancer Detection

Dr.Li mentions the procedure of current cancer detection,including extracting DNA from blood cells, amplifying it with PCR and then getting its sequences.Detection varies from diseases and gene chip(detection for all genes) also could be applied with enough money.However, only some genes are of necessity to be detected for individuals.

(2)Gap Exists Between Clinical Application and Basic Research

There was distance between clinical application and basic research in early detection of cancer. For instance, some means of early cancer detection still had a long way to go before proved efficacious---commonly 5-10 years experimental validation phase is integral to clinical application.

(3)Stability of MiRNA in Blood is Doubted

Dr.Li shows her concern about the instability of miRNA when the topic is involved with miRNA and its potential application in early detection of cancer.However,she also mentions that as a clinical doctor,she doesn’t have much research time so that she is not expert in miRNA field.Meanwhile,Dr.Li says that they clinical doctors usually pay more attention on developed technology instead of the developing one and her knowledge about miRNA comes from papers.

(4)Efficiency of MiRNA Detection is Not Guaranteed

Dr.Li says that they clinician commonly assess the severity not only by the gene detection,but also by patients’ clinical symptoms,chromosome. The oncologists even need to do the Immunohistochemical checker and pathology section to make sure.So they suspect a bit about the efficiency of early cancer detection due to the lack of pathological symptoms and currently so-so specified biomarkers in patients’ early stage.

(5)Prospect of MiRNA Is Expected

Dr.Li hopes that the team of NUDT_CHINA would find a way to detect cancer, with much greater specificity and fewer side effects, so that more targeted medicine could be invented to kill cancer tumors. She also figures that early cancer detection could help people carried with household hereditary disease factors.Additionally, the index of miRNA could help alert people to pay more attention to their living habits and health condition as well.

2.Dr.Hou Tao, Oncology Department，Second Xiangya Affiliated Hospital of CSU

(1)Current situation of tumor biomarker test

We asked Dr.Hou about some details about current tumor biomarker test the hospital usuallly suggested patients to take. And this helped us learn more about the current situation of tumor biomarker test.

a.Biomarker tests in health exams

In a normal health exam, there is an optional choice of tumor biomarker test including tests for 12 biomarkers of most frequent cancers, which is called ‘set 12’ by doctors. Talking about taking the biomarker tests, Dr.Hou said that the popularity of biomarker tests was increasing in oncology department in hospitals in China. And with more people getting familiar with tumor biomarker test, it was gaining increasing attention, and a great number of healthy people requested tumor biomarker test in their health exams, especially the elder. Tumor biomarker tests are gaining increasing attention from the crowd.

b.Details of a normal biomarker test in health exams

Contents of ‘set 12’ biomarker test in a normal health exam:

CA19-9	Beta-HCG	CA125
NSE	AFPHY	Ferritin
CEA	Free-PSA	HGH
CA242	PSA	CA15-3

Cost of the test for 12 biomarkers, or the ‘set 12’ is ￥500, including biomarkers of breast cancer, liver cancer and so on. Dr.Hou also told us that the validness of biomarkers was not so optimistic, thus could only work as a reference for diagnosis of cancers.

(2)Advice for The Project---SPLIT-GFP, SPLIT LUCIFERASE, OR SPLIT HRP?

To improve the sensitivity and specificity of RCA output signal and to visualize the RCA outputs in the wet-lab experiments, we designed a single guide RNA mediated dCas9 binding system and a conjugated reporting system. However, dilemma came that split-luciferase, split-HRP and split-GFP all seemed to meet our requirements of reporting the signal. Which was the best fit?

Firstly we searched for information of the three reporting systems:

	Color reaction principle	Requirements for color reaction
HRP	Catalyze TMB to change color	Protoheme as cofactor
GFP	Absorbs light at a certain length and fluoresces	Light at a certain length as motivator Fluorescence microscope
Luciferase	Catalyse luciferin and make it fluorence	Fluorescence microscope

Then we turned to our medical consultant Dr.Hou and asked him about suggestions of our reporting system.

We analyzed the situation together, had a talk about our novel blood-microRNA detection system, and discussed the disadvantages of current early cancer detection methods as well as expected new points of our project. Finally this helped us decide which reporting system we would use---the split-HRP.

Survey In The Hospital & Health Center

We did surveys in Second Xiangya Hospital affiliated to CSU and Health Center of NUDT about details of current early cancer detection methods, which acted as a reference to our project. Here is a summary.

METHOD	COST (RMB)	TIME	ADVANTAGE	DISADVANTAGE	POPULARITY
PET-CT	7000-8000	2-3h	Detection for early and advanced tumor No pain Valid	Expensive Rare Radiation Condition requirements (not fit for remote  areas)	270 in the whole country (data source:2011-2015 National PET-CT Plan by Chinese Department of Health)
CT	200-500	20min	Most effective for tumors in nervous system, head, chest and abdomen	Only functions on a certain part Radiation Condition restriction	Rare in counties and remote areas
Tumor biomarker test	500 (including 12 biomarkers)	10min (sample gathering)	Easy to operate No side effects	Requirements for instruments Low validness Narrow testing range	City or central hospital

Conclusion drawn from the result:

a.Costs of current early cancer detection methods vary largely, but on the whole the price is not low, which may make it hard for poor families to afford. Plus, it may also turn down people’s positivity to take early cancer detection.

b.These test methods have high need of good external condition, eg. Electricity, instruments, operation environment. These may restrict the early cancer detection in big cities or counties, leaving remote area aside.

c.Popularity of these test methods is low, which we can see from the result that people almost can take early cancer detection only in developed cities or counties. This makes it hard for people in remote areas, such as rural districts and behindhand villages, where cancer occurrence rate is also high and cancer death rate is even higher because of backward medical services.

From these we can tell that a novel early cancer detection method that is more convenient, cheaper and has less requirements for environment is in great need. And this is exactly what our project this year is striving for.

Sample Collection for Wet Lab

Since our project this year was to develop a blood-microRNA detection system as a novel method for early cancer detection, and results of previous wet lab all confirmed the effect of our detection system, it was necessary to apply it to serum as a final confirmation. We came to the health exam center of Affiliated Hospital of NUDT and talked with doctors and 5 non-small cell lung cancer(NSCLC) patients about our need for serum samples to and got their support and permission. Also we had 30 healthy postgraduates from NUDT as our volunteer of serum samples. And we collected the blood samples with the help of doctors.

1.Approve of the Informed Consent Form

We submitted the Informed Consent Form to the department of ethics in NUDT. After days of checking it was approved to use.

2. Consultation with doctors

Firstly we found Dr.Tan, one of the principles in the department of clinical lab of the health center. He introduced the formal process of collecting blood samples to us and approved our Informed Consent Form. Dr.Tan also helped us with the collecting work.

3.Meeting with sample groups

We talked to 5 cancer patients in different stages of NSCLC as well as 30 healthy volunteers. We introduced what we had been working on and explained what we would do with their blood samples. We also made a security guarantee that we would keep their personal information as secret.

4.Processes of sample gathering

a. Determination of sample group of patients and healthy volunteers and register their information.

b. Filling in Informed Consent Form and diagnostic information.

c.Doctors collected 5L blood sample/person with coagulation tube and match each with the person’s information(including name, age and extra information as AD, stages of NSCLC, etc from cancer patients).

d.Samples transmitted to professional operators and serum was separated.

e.Registering samples in the information management system and setting up links with the Informed Consent Form.

f.Sample serum was transmitted to cryopreserve tubes and placed in -80℃ in the lab.

PART II

PRODUCT EVALUATION

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Market Demands

Previously we did surveys in hospitals and health center about current early cancer detection methods. To gather more information, we searched the Internet about the same issue. Here is the summary.

Looking at both results we found that the biggest disadvanges of current early cancer detection were costs and side effects. And this informed us of the direction our project should strive for---handy and low cost. Thus we made a cost evaluation for our product-to-be. Here are the results.

Cost Evaluation

We made an evaluation of efficiency and price of our detection system in this part to see how much our product may decrease the cost of biomarker detection for cancer, or to say how much our project could help the world with.

The other reason we made this cost evaluation was to make it a feedback for the project and find other aspects it may develop itself in.

The following is the evaluation of our expected product cost.

Click here to view details of contents, instrument, reagents and experimental procedure of our product-to-be.

Project Contents

miRNA extraction; Incubation from protein and sgRNA; Fluorescence coloration

Major Instruments and Reagents

Instruments

Centrifugal machine, Freeze drying machine, 37 degree constant temperature box, Filter paper and other laboratory supplies, Computers and printers

Reagents

Ribonuclease inhibitor, Double distilled water, Special probe , Phi29 DNA polymerase, Buffer phi29, BSA, dNTP, Cas9 protein,and sgRNA

Experimental Procedure

Firstly,freeze-dry the required reagents (such as enzymes, proteins, buffer, etc.) in a solid carrier (filter paper, etc.).

Then,get serum from provided blood, boil to destroy binding between miRNA and protein and extract miRNA.

Finally,add extracted miRNA to the solid carrier and wait for coloration,compare with the standard color card to estimate contents of miRNA.

1.Cost Evaluation

name of reagents	Unit price	Use level	Equivalent
Ribonuclease inhibitor	$32.2/200ml	37.6ul	$0.006
phi29 DNA polymerase	$70.5/250U(10U/ul)	0.6ul	$1.692
Buffer phi29	$19.5/5ml	3ul	$0.012
dNTP	$12.0/1ml	7.2ul	$0.086
Special probe	$0.16	one	$0.163
Cas9 protein	$22.3/g	1ul(0.2uM)	$0.713
double distilled water	$3.0/25kg	491.2ul	$0.000

Assuming that the protein can be made to the level of industrial production, the total reagent cost is about $2.672.

Name of equipment	Business	Price
centrifugal machine	TGL-16 high speed centrifuge (1.5ml*12 hole)	$77.979
Freeze dryer	LY-15AC high efficiency freezing and drying machine	$450.224
Constant temperature box	DH-250AB electric heating constant temperature incubator	$335.908
HP printers	HP color laser integrated machine	$427.382

All of these machines can be used for long and we don’t need to take them while testing miRNA outside. So we consider loss only. Centrifugal machine is used to extract serum while freeze dryer is used to freeze-dry relative reagents in the test paper. We apply HP color laser integrated machine to make colorimetric card.Briefly,the loss price is about $0.3.

2.Interpretation of Result