🎉 JOGL is soon launching a new version. All the users of the v1 will be migrated to the new version. In the time being, we do not allow the creation of new users on this platform.
Magnus-qPCR: An open, portable and low cost qPCR machine. banner
Project
5
Members

Status:
Active/Ongoing
Project maturity:
Prototype
Linked to group(s)/challenge(s):

Magnus-qPCR: An open, portable and low cost qPCR machine.

About reviewed project
The qPCR technology allows us to precisely quantify DNA/RNA present in samples. This is important in diagnostics (eg. Covid-19), to study the an organism metabolism by measuring the gene expression and to compare point mutations (SNPs).

Magnus-qPCR

An open, portable and low cost qPCR machine.


While qPCR is one of the most used tools in molecular biology laboratories, this equipment is usually large and expensive (usually costing tens of thousands of dollars), restricting not only laboratories with few resources, but also geographically, making research and diagnosis in places impossible remote, even for researchers with many resources. This problem is even worse if we think that many samples to be analyzed are perishable, creating a logistical challenge.


If we take into account that it is a diagnostic tool and that many pandemics are yet to come, it would be a dream if this technology were so widespread that ordinary people, with little training, could decide whether or not they can leave the house before they infect somebody. Although this scenario is utopian, we intend to create a machine that would fulfill the requirements for this: portable, accurate and inexpensive. So lets meet Magnus-qPCR, an affordable real time PCR machine!


Specifications*:

  • 8 samples
  • 1 to 4 channels, you can customize it! (FAM/Sybr Green,HEX,ROX/CalRed610,Cy5)
  • MicroSD card slot for data logging
  • Capable of High Resolution Melting Analysis for SNP detection
  • WiFi interface, so you can control and visualize data from a smartphone/PC
  • Portable (less than 150 mm in size)
  • USB powered (5V 3A power supply)
  • CHEAP


*This machine is a work in progress, so the specifications may change and/or be innacurate. Also, don't try to replicate it if you don't know what you're doing, as it probably won't work until we release a stable version.


How will it work?

The machine can be roughly separated into 3 parts:

  • The main PCB, containing the microcontroller and the heater;
  • The PCB of the reading head, containing LEDs and light sensors;
  • The optical reading head, which will house lenses, beam splitters, filters, prisms, etc., as well as the stepper motor responsible for positioning this optical assembly above each tube;


1.0 Introduction

    The world has been suffering from the Covid-19 pandemic for more than a year. The situation is so critical, with new variants emerging at the same time that vaccines are being tested, that even developed countries, with good health systems, are being severely affected.


1.1 Problem and Background


Tests are essential for the efficient control of the pandemic, as they allow to monitor the progress of the disease, the appearance of outbreaks, border control, accurate diagnosis for supposed patients. Unfortunately, something so basic has been suffering from scarcity, both by reagents and by equipment. The most accepted tests today use RT-qPCR or RT-LAMP technology. The RT-qPCR test consists of measuring the concentration of the virus genetic material present in a patient sample. As this genetic material is too diluted to be detected, the equipment tries to multiply this material. More specifically, it tries to multiply certain genes unique to the coronavirus. A dye that is also introduced in the reaction is responsible for showing how much of these genes are present, as it will fluoresce if the viral genes have been amplified. The RT-LAMP test works in a similar way, but uses different reagents and the detection mode is more flexible, not just based on fluorescence. Unfortunately, equipment capable of carrying out these tests generally costs tens of thousands of dollars and is large, restricting them only to large research centers.


1.2 Solution summary in simple terms


We propose to build a low-cost (~$70 up to ~$150), portable, open source qPCR machine, flexible enough to be compatible with most tests and supplies on the market.


1.3 Solution summary in technical terms


The most common tests using RT-qPCR technology use the SYBR Green fluorophore (At ~ 520 nm), or in multiplexed tests, fluorophores such as FAM, HEX, ROX and Cy5, which emit at 520 nm, 555 nm, 610 nm and 670 nm respectively. Multiplexed tests have the advantage of detecting multiple genes in a single tube / reaction, speeding up the process and the machine's capacity. The tests generally need a positive and a negative control, so two wells will be used for this purpose. In order to maintain the portability of the machine, we decided on the number of 8 wells, so it will be possible to run 6 tests simultaneously. The machine will have 4 channels, and the user can choose which channels he will want. The one-channel version will cost about $70 to manufacture and the full 4-channel version will cost ~$150.


1.4 State of advancement of the project


We made some prototypes using generic components (heaters, lights, sensors) to be able to develop the software that will run on the machine. This software works as planned, and we can run PCR cycles uninterruptedly, recording the data on disk. We built some heaters similar to those that will be used in the final version of the machine, in order to validate them experimentally, as they are an innovative and critical part of the machine. We also designed all the hardware side by side with what is available on the market, with no need for customization, with parts widely available online. We have a spreadsheet with all suppliers and quotations, so that as soon as we have funds, the parts will be quickly purchased to assemble the equipment.


1.5 Project Timeline


Upon receiving the funds:

  • Purchase of optical components and PCBs

 

After receiving the purchased materials:

  • Adaptation of our software to the real machine
  • Machine detection limit tests
  • Homogeneity tests between wells
  • Stress tests
  • Tests with positive controls
  • Distribution



2.0 Project Implementation


2.1 Solution, research, or intervention?


This equipment is a solution found for the shortage of low-cost and portable machines on the market. Where we live (Brazil), tests are done only in large research centers and laboratories, which leads to centralization. This leads to logistical problems, slowness, price increases and underreporting of cases.


2.2 Methodology


    For the construction of the equipment, in our GitHub there is the entire project available, in case anyone wants to replicate it. There are optical components and PCBs to be purchased and parts to be printed in 3D.

   For the tests to be done:

Test of detection limits: a standard curve will be made using a serial dilution of fluorescein, or a qPCR reaction will be made with different concentrations of DNA template, with probes for each fluorophore that the machine is able to detect.

Homogeneity test: using data from the previous experiment to find the sensitive region of the machine, we will run qPCR in all wells of the machine using the same concentration of DNA template.

Stress test: we will run tests for days uninterrupted and check any changes in specifications.

High Resolution Melting: using samples with different numbers of SNPs (single nucleotide polymorphisms) and slowly increasing the temperature, we will check if the machine is able to distinguish between these sequences.


   


3.0 Safety, quality assurance and regulation


3.1 What steps have you taken to ensure your solution’s safety? How advanced are you in this process?


    Initially we do not need to test the equipment with samples from real patients, the positive controls can be plasmids with the genes that the tests use (usually ORF1ab, gene N and gene S), so we will not be in contact with potentially contaminated material, being necessary only a BSL-1 laboratory for the manipulation of plasmids. We contacted biotechnology companies, which received us very well and offered us donations of detection kits and support.


3.2 Have you planned the conduct of your manufacturing process that ensures quality, what are the steps you have taken? How advanced are you in this?


We will not manufacture the parts, we will send the end users a box of parts to assemble the machine, with an assembly manual, which will be easy and uncomplicated. All parts will be purchased from major suppliers in China.


3.3 Will you need assistance with the regulation system? If not, which regulatory system do you plan on using to distribute the product?


Initially, this equipment can be used to assist researchers in their experiments in the laboratory or in the field, but for medical use, we will need regulatory assistance.


3.4 Have you talked to medical staff about the feasibility of your project? What did they say?


    We contacted test and enzyme manufacturers and the feedback was great. In addition to being interested as end users, they are willing to help us with development as well.


3.5 Have you planned the testing, verification and validation of your solution? How advanced are you?


We validate the software, which works as expected. We need more funds to purchase parts in order to have real equipment and test it.


4.0 Impact, issues and risks


4.1 What impact do you feel your project could have?


We hope that it will be used to check the presence of the virus in environmental samples (such as sewage and air) for better management of the pandemic, to assist researchers in research (such as analysis of gene expression) and, if possible, diagnostics.


4.2 What do you think would make your project a success?


Our equipment is innovative in terms of cost (the most basic version costing ~$70), portability and flexibility, while being conservative in terms of technology as qPCR and LAMP are already well known and accepted. We believe that this combination will be crucial for the success of this project, and we see this due to the good acceptance of the market.


4.3 Please list the known issues, potential risks, grey-areas, etc in your project


We need to learn more about the regulatory process for building diagnostic-related equipment. For the research area it will be easier to implement the machine.



5.0 Originality


5.1 What other projects on JOGL are like yours?


We found Ninja-qPCR a similar project. There is also OpenEnzymes, which seeks to reduce the cost of enzymes needed in tests, which complements our project.


5.2 Is this an innovative project? What makes this project different if it’s unique on JOGL?


   Although Ninja-qPCR is a project similar to Magnus-qPCR, its approach is different, as the former can process more samples simultaneously, while Magnus-qPCR have less wells but more flexibility. Also we are geographically distant, so it makes sense to have two different machines. We also plan to expand our machine to include Raman spectroscopy, since the optical system is similar.


5.3 Is there already an open source version of this project?


There are some similar projects, like the Chai qPCR, but it is not portable like ours and costs ~ 100x more than our machine.


6.0 Team experience


6.1 Please cite your team members and their roles in the project.


Felipe Tanaami: He studied physics at Unicamp, although he spends all his time in the area of molecular biology and mechatronics. He has been developing low-cost laboratory equipment for years. He also has experience in wet lab.


Isabella Messuti: He has experience in the cultivation of fungi, plants and wet labs, in addition to being formidable in PCB design in KiCAD and Inkscape.



7.0 Funding and Costs


7.1 Please provide a costing of your project be as detailed as you can


A spreadsheet with costs can be found at this link.


7.2 How is your project being funded so far?


Until now, we were developing software and hardware that requires little investment. But with the testing phase, we need more funding than we can provide. The tests we have done so far have been with our own resources, including expenses on electrical components, parts and molds for heaters.


7.3 How much funding do you need and how do you plan to use that funding?


The testing phase is one of the most costly as it requires several materials for prototyping, in order to be able to certify the effectiveness of our equipment and obtain solid data about it. This includes: sensors, LEDs, filters and lenses for the optical system; dry film, copper plates, or PCBs printed on the JLC for testing with the heater and 3D printed parts to house the optical system. In addition to the tests, the money will also be used for the final production of the equipment, which will cost around US$150 for the 4 channel model and around US$70 for the simplest model.


The minimum quantity that PCB manufacturers accept is 5, so we base it around that number. To build 5 machines we would spend about 950 dollars, including freight. If we can buy a 3D printer, that would be another 300 dollars. Therefore, for 1250 dollars we would be able to have 5 pieces of equipment and the means to manufacture and prototype the optical assembly. Some reagents will be needed for the tests as well.


We calculated that if we had $ 1,500 available to invest in this project, they would be well spent for the development of the equipment.

Additional information
  • Short Name: #MagnusqPCR
  • Created on: March 5, 2021
  • Last update: July 12, 2021
  • Grant information: Received €1,131.30€ from the OpenCOVID19 Grant Round 5 on 03/24/2021
Keywords
Biotecnology
Hardware & software design
Front-end development
3Good Health and Well-being
6Clean Water and Sanitation
14Life Below Water
15Life on Land