martes, 25 de noviembre de 2014

Open qPCR: DNA Diagnostics for Everyone

Turning DNA into data just became affordable, and biohacking will never be the same. Meet our open source Real-Time PCR Thermocycler.

Real-Time PCR is a powerful technology. It can detect foodborne contaminants like E. Coli and Listeria, as well identify fraudulently labeled food products. It's used to diagnose infections such as HIV and Malaria, and we've created a reward level to donate an Open qPCR to Ebola clinics in Western Africa.

Real-Time PCR can also identify genetic mutations that increase our likelihood of cancers, such as those within BRCA1. It's a fundamental tool of biological research, and has literally thousands of other applications too long to list here.

However with machines costing $20,000 and up, Real-Time PCR (also known as qPCR) is simply unaffordable where it is needed most. We aim to change that with Open qPCR, a system available at a fraction of their typical cost.

Four years ago we Kickstarted and successfully delivered OpenPCR, the world's first open source thermocycler. Now we're taking it to the next level with a machine that not only copies the DNA but converts it into data.

Open qPCR is disrupting DNA diagnostics.
Our reason for starting this project is simple: we want to make this essential technology available to everyone, including doctors in developing countries, students in high school and university labs, companies in the food supply chain, and biohackers who are developing some of the most innovative synthetic biology applications.

Over the past two years, we've designed and prototyped an open-source instrument that can perform the above mentioned tests, but costs less than a tenth the cost of other commercially available systems. Now, we're ready to share it with the world and see what others can do with it.

DNA Diagnostics on your Desktop
To create a truly cost-effective diagnostic system, we realized users would also need affordable access to the necessary reagents. That's why we created our own low-cost Real-Time PCR MasterMix incorporating our open source fluorescent dye.

We also focused on making the software as user-friendly as possible. While there's powerful functionality available for scientists, the machine also interprets the data and presents clear positive/negative results to end users.

Open qPCR web UI showing detection interpretation. Results are labeled indeterminate if replicates vary or control reactions fail.
What Open qPCR Can Do
The Hardware
Open qPCR's heatblock holds 16 reaction wells capable of holding either 100 or 200 uL tubes/strips. The heat block ramps between 0 and 100 degrees C at 5 C/s, allowing many PCR protocols to be completed in as little as 30 minutes.

Open qPCR's well block supports 16 samples

Fluorescence detection is performed with a solid-state LED excitation (470-490 nm) and photodiode detection system, utilizing a set of optical filters to create sharp cutoffs.

Open qPCR is available in both single-channel and dual-channel versions. Both versions can detect many green fluorophores like FAM in the 510-545 nm range. The dual-channel version can additionally detect HEX/VIC/JOE fluorophores in the 560-610 nm region.

The optical system detects the DNA in the right tube, but the left tube without DNA fails to fluoresce. The single-channel detection system is shown in this image.

An 800x480 pixel capacitive touchscreen allows easy control of the machine, while USB, ethernet, and wifi interfaces let the user control the machine via a web browser or other networked systems. The machine is controlled by an embedded BeagleBone Black microcomputer, running embedded Linux at 1 GHz and sporting 4 GB of flash.

Open qPCR uses ethernet, wifi, and USB to communicate with the outside world

Open qPCR is compatible with power systems around the world (110 - 240 V, 47-63 Hz), and complies with all CE and FCC requirements. In normal cycling operation the machine requires 250 W, but in a special low-power mode, isothermal DNA detection can be performed with as little as 45 W, allowing field use with solar/battery power.

The hardware design, including BOM, SolidWorks files, and Eagle CAD will all be released as open source when the machine ships, and are available sooner as an early access reward.

Tech Specs

The Software
We've developed an open and intuitive user interface using modern web technologies such as HTML5 and JavaScript. Open qPCR runs an embedded web server, which lets the machine be operated in a platform-independent way from nearby computers connected via ethernet or wifi. A USB connectivity mode is also supported for non-networked environments.

Open qPCR provides a powerful protocol editor, which supports basic cycle and step editing, as well as advanced features such as controlled ramp rates and auto-delta/touchdown steps. Data collection may be triggered on each step and/or ramp, and a final hold temperature is supported.

A visual plate layout editor allows the assignment of samples, targets, standards, and controls to each well, as well as viewing fluorescence curves on a per-well basis.

The software supports amplification curve Ct thresholds, presence/absence detection, melt curve analysis, and relative quantification. Software support for absolute quantification may be ready by the shipping date, or will otherwise be made available shortly thereafter as a free downloadable update. It's a bit tricky to do absolute quantification with a 16 well block, but we've got some ideas. Data may also be exported in RDML format, allowing for more advanced analysis in other software.

All software including real-time control, web user interface, and scientific analysis code will be released as open source when the machine ships.

Real-Time PCR Reagents and Starter Packs
Real-Time PCR reagents have a reputation for being expensive, which is why we're creating our own to coincide with the low cost Open qPCR. We've created our own low-cost PCR MasterMix, and are currently synthesizing Chai Green, an IP-free fluorescent intercalating dye. The composition of these reagents and structure of Chai Green will be released as open source.

We'll be including starter packs of these reagents with all Open qPCR machine rewards so you can evaluate these low-cost reagents yourselves. The starter packs will include enough reagents for 50 PCR reactions.

Of course you may also use existing dyes or probes with fluorophores like FAM with your Open qPCR.

Our Story
Four years ago, in 2010, Josh Perfetto and Tito Jankowski started a Kickstarter campaign for OpenPCR, the world's first open source PCR thermocycler. Josh and Tito successfully fulfilled the Kickstarter, and today approximately 800 OpenPCRs are used around the globe.

Though revolutionary at the time, OpenPCR, like all endpoint PCR thermocyclers, was a relatively basic machine. It simply heats and cools a piece of metal in a precise manner. This thermal cycling facilitates the PCR reaction which selectively amplifies DNA, enabling the many applications of PCR described on this page. However turning this amplified DNA into useful information requires downstream laboratory processes which are too laborious, costly, and error-prone for most diagnostic uses.

This provided the impetus to create Open qPCR, a machine that directly turns DNA into actionable information. In 2012 Josh assembled a team of electrical, mechanical, optical, and software engineers to couple the thermal cycling accuracy of OpenPCR with an optical detection system, powerful modern processor, and modern ethernet, wifi, web, and touch screen interfaces. And unlike OpenPCR, which was a build-it-yourself DIY kit, Open qPCR is a professionally assembled ready-to-use system, though it stays true to its DIYbio roots.

It was a long haul, but we're excited to bring it to you.

The Biohacker Kit
To perform tests with Open qPCR, beyond the machine and provided reagents, you'll need a set of PCR Primers specific for your application and control DNA to have confidence in your results. We will be creating an open source database of PCR assays (the primers, probes, protocols, and controls needed to perform PCR) in 2015, and early beta access to that is available as a KickStarter reward.

However as a first step towards that vision, we're offering a Biohacker Kit available separately or as an add-on to an Open qPCR reward. The Biohacker Kit ships internationally and contains:

  • PCR primer and control library for 5 tests: one pertaining to fraudulently labeled food, another to a gene associated with athletic ability (ACTN3), and three other tests which will be decided on by backers
  • Reagents for 50 DNA extractions
  • Real-Time PCR reagents for 100 reactions
  • A laboratory pipette
  • Two boxes of pipette tips
  • PCR strips
  • Sterile cotton swabs for collecting DNA
With our machine and this kit, you'll have everything you need to perform these tests right out of the box.

What's in the Biohacker Kit
Production Schedule
Over the past two years we've built and tested countless prototypes. We now have a working system, and have suppliers and manufacturers lined up to build our machine. We're ready to get Open qPCR into production, but need your help to finance the first manufacturing run.

We plan to have the first batch of units to fulfill the Kickstarter rewards in March 2015, and the second batch ready in April. The expected delivery date for your batch is shown in the reward section when you pledge.

We are continuing to refine the software, and are on-track for basic informatics (amplification curves with Ct values, melt curves, and presence/absence detection) to be fully implemented by our shipping date as well as relative quantification. Support for absolute quantification is on our development roadmap, but may or may not be ready by the shipping date (it's somewhat tricky with our 16 well block). If it is not ready in time, it will be available as a free downloadable software update shortly thereafter.

Progression of Open qPCR's circuit board

Open qPCR has not yet been submitted to nor cleared by the US FDA for medical uses. Thus within the US and EU it is sold for research, educational, and recreational uses only. We will ship Open qPCR globally but you are responsible for ensuring your intended uses are compatible with the regulations of your country.

Add Ons
The T-Shirt, Coffee Mug, and BioHacker Kit rewards may all be added to the higher reward levels by adding the cost of these rewards to your total pledge. If you are shipping outside the US, please add a $15 shipping fee for T-Shirt or Coffee Mug add-ons, and a $60 shipping fee for BioHacker Kit add-ons. You will be able to specify the add-ons you are requesting in a post-campaign survey.

A reagent-only version of the Biohacker kit is also available for $150, plus $40 for shipping outside the US. This kit includes the DNA extraction buffer, Real-Time PCR MasterMix, primer library, and control DNA of the Biohacker kit, but does not include the pipette, tips, cotton swabs, PCR tubes, or eppendorf tubes.

About Us
Chai Biotechnologies is a biotech startup focussed on making biotechnology more broadly accessible. Chai is based in Santa Clara, California, and was founded by Josh Perfetto, a former co-creator of the OpenPCR open-source thermocycler project KickStarted in 2010. Chai is a team of 8 including experts in molecular biology, mechanical and electrical engineering, software development, and user interface design.

We have worked to remove as much risk as possible before launching this campaign. We have created a working prototype, sourced all key components from suppliers, and identified a local manufacturer for our initial batch, which will help us resolve manufacturing issues quickly. Additionally we have successfully delivered on an earlier Kickstarter project OpenPCR, which was a simpler predecessor to this machine.

While we're happy with our preparations to date, there is always the risk of unforeseen challenges when manufacturing physical products. We will keep you advised of our progress and any unexpected challenges, and will work our hardest to meet the delivery schedule.

ORIGINAL: Kickstarter

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