BioCoder #6

Chapter 4. Open qPCR: Open Source Real-Time PCR and DNA Diagnostics

Josh Perfetto

In November 2014, we launched a Kickstarter campaign for Open qPCR, which raised $202,701 and dramatically lowered the cost of a general-purpose real-time polymerase chain reaction instrument (such devices typically sell for more than $20,000). More importantly, we just open sourced a technology that is the gold standard for diagnosing viruses in humans, detecting pathogens like Salmonella in food, genotyping many genetic variants, and a myriad other applications. While the first Open qPCRs are being manufactured this winter, I wanted to take a moment to explain real-time PCR and its benefits.

Let’s start with basic PCR. Suppose we want to know if a patient is infected with malaria. Detecting the malaria parasite in a patient’s blood is like finding a needle in a haystack. To do so, we first need to amplify the malarial signal above the background noise, which is exactly what PCR does.

All of the DNA extracted from the patient sample goes into the reaction, and only a specific target DNA sequence is selectively copied. PCR is a chain reaction of typically 30–40 cycles, and each cycle of the reaction doubles the amount of target DNA present. Thus, if malaria DNA is targeted PCR exponentially amplifies this DNA to detectable levels, but if and only if the malaria DNA was present to begin with. Facilitating this chain reaction requires the repetitive heating and cooling of the reagent/DNA mixture, which is what a basic “endpoint” thermocycler such as OpenPCR does. However, with these endpoint machines, you’re left with a tube of DNA as your output; you don’t know whether the patient has malaria without performing additional laboratory work.

Real-time PCR takes things one step further by integrating an optical fluorescence detection system into the instrument. This lets the instrument detect the amplified DNA, and thus provides information rather than DNA as an output. In a process executed by a single device, you can determine whether the patient has malaria and see the result on a computer interface.

At this point, the astute reader of BioCoder may be saying, “Aha, but I could use a basic thermocycler and then run a gel!” To an extent you could, but if you went to your doctors and had a blood test run for a suspected virus or pathogen, you could be virtually assured that the lab was not running a gel. Let’s examine the benefits of real-time PCR and why it’s often considered to be the gold standard for diagnostics.

Benefit #1: More Specific Detection Reduces False Positives

The specificity of the basic PCR reaction depends on two DNA primers annealing with their complementary sequence. However, such primers can frequently “misprime” and anneal elsewhere, amplifying an unintended sequence of DNA. Many real-time PCR diagnostics utilize an additional fluorescently labeled “probe” DNA that must anneal to a specific sequence within the amplified segment for a signal to be detected. In the event that the two PCR primers misprimed, it is highly unlikely the probe would find a complementary sequence within the incorrectly amplified sequence. Other real-time PCR tests don’t use probes, but measure the melting temperature of the amplified sequence to ensure it is the correct one. Both techniques help avoid false-positive results.

Benefit #2: Internal Positive Controls Reduce False Negatives

In any PCR test, you need to run a positive control: if the positive control doesn’t amplify, you can’t interpret a sample’s failure to amplify as a negative result. However, with standard PCR, this positive control is run in a different tube, meaning it’s possible for the control reaction to work but the diagnostic reaction to fail for some other reason (pipetting error, PCR inhibition, etc.). With real-time PCR, the control and diagnostic reactions can occur in the same PCR tube, and the results can be differentiated using different optical channels. This is known as an internal positive control, and it eliminates a number of problems that can cause false negatives.

Benefit #3: Quantitative PCR (qPCR)

In many applications, such as analyzing gene expression or measuring viral load, it is necessary to not only detect but also quantify the amount of DNA present. With endpoint PCR, you make a fluorescence measurement only after the entire PCR run is complete, and you typically detect either a whole lot of DNA or none at all.

With real-time PCR, fluorescence measurements are made after each PCR cycle. During the first several cycles, no signal is observed, but eventually a weak, then strong, and then saturated signal will be seen. A real-time instrument uses not only the level of signal observed but also the cycle number of when it was observed, to quantify the amount of DNA present (thus the term qPCR). For example, suppose a certain signal level of sample A is seen at cycle 15, while the same level is not seen in sample B until cycle 18. Assuming 100% efficiency, you could conclude that sample A contained eight times the starting DNA as sample B, as the target DNA present doubles each cycle (in practice, you would measure the efficiency rather than assume it, and the instrument would employ a much more sophisticated calculation).

Benefit #4: Saves Time and Money

A standard endpoint thermocycler takes in DNA and gives out DNA. Obtaining a diagnostic result from that output DNA requires further downstream processing, such as casting and running a gel. Doing so requires time and reagents, which of course equals money. Once you have a real-time PCR thermocycler, the per-sample cost is actually quite cheap, and you can get diagnostic results in as little as 30 minutes.

Benefit #5: Genotyping

A common use of real-time PCR is for genotyping, or determining the specific allele of a gene present. PCR primers can commonly be designed to amplify a region of DNA that contains the variants of interest. But once it’s amplified, how can you identify the internal sequence?

In genotyping applications, there are commonly a small number of potential sequences present, and these sequences will have different melting temperatures (the temperature at which the double-stranded DNA dissociates into two single strands). A real-time PCR instrument can detect which variant is present by doing a melt-curve analysis: the temperature is slowly increased while the fluorescent signal is monitored. As the fluorescent signal is proportional to the amount of unmelted double-stranded DNA present, the melting temperature can be determined.

Open qPCR

We’ve talked of the many advantages of real-time PCR but neglected the typical disadvantage: cost. Real-time instruments typically cost $25,000 to $50,000, which greatly restricts access to this technology. With Open qPCR, we set out to reduce the cost by an order of magnitude and deliver a modern web-based interface that makes the device easy to use.

Open qPCR is a real-time PCR thermocycler that processes 16 samples at a time and ramps the temperature at 5 degrees Celsius. Such fast ramping speeds enable fast run times: test results can be obtained in as little as 30 minutes. The device employs a solid-state optical detection system consisting of LEDs, photodiodes, and optical filters. Open qPCR is available in both a lower-cost, single-channel version, and a dual-channel version that can detect multiple fluorophores at the same time and thus utilize internal positive controls.

Open qPCR is built upon the Beaglebone Black development platform, which runs embedded Linux on a 1 GHz ARM processor. The device has Ethernet, WiFi, and USB connectivity. We also included an embedded touchscreen interface, so the device may be used in the field without any PC connectivity.

Ease of use depends a lot on the software, so we spent a lot of time on user-interface design. Open qPCR runs an internal web server to deliver an HTML5/JavaScript interface, which can be accessed via any computer or smartphone. This software allows the user to graphically edit the PCR protocol; define the plate layout; and view amplification curves, standard curves, and interpreted diagnostic results. An open REST API allows Open qPCR to be controlled by external automation systems.

Open qPCR is also completely open source: the mechanical and electrical CAD designs, real-time control software, scientific analysis software, and web interface will all be released as open source when the machine ships in March 2015.

Our eventual goal is to make reliable DNA diagnostics available not only in the lab, but to all who have innovative ideas on how to apply this technology: beekeepers with a theory on population decline, consumer activists fighting fraudulently labeled food, and educators teaching a new generation of bioliterate students. The launch of Open qPCR is a first step, but there’s far more to come to complete this vision.

Josh Perfetto is the founder of Chai Biotechnologies, a Santa Clara, California–based start-up creating modern tools for molecular and synthetic biology and deploying the power of these technologies outside of the lab. Prior to founding Chai, Josh led engineering teams in a number of software and wireless start-ups, and in 2010 he cofounded OpenPCR, creators of the world’s first open source PCR thermocycler. He can be reached at [e-mail] or @jperfetto. Learn more about Open qPCR at www.chaibio.com/openqpcr.