By Ben Butkus
GÖTEBORG, Sweden – TATAA Biocenter held its European qPCR symposium, "Developments in Real-Time PCR: Research and Molecular Diagnostics," at the Swedish Exhibition and Congress Center here this week.
PCR Insider was there to cover the symposium. Following are notable news items from the conference floor.
DiaGenic Takes PCR-Based Tests to Market
Anders Lönneborg, research director for Norwegian molecular diagnostics outfit DiaGenic, provided an update on his company's quest to market quantitative real-time PCR-based blood tests for Alzheimer's disease and breast cancer.
DiaGenic has been developing its tests, called ADTect and BCtect, for several years. The tests are based on the idea that the "primary diseased part of the body is not the only part responding to disease" and that there is a "subtle but systemic impact on gene expression" that leaves a unique signature in the body, Lönneborg said during a presentation.
As such, ADTect and BCTect examine a panel of the most important genes, covering a wide array of biological processes, which change in response to the early stages of Alzheimer's or breast cancer.
DiaGenic considered both DNA microarrays and qPCR arrays to run its tests before ultimately deciding on the PCR-based platform. After whittling the number of genes examined in each test from tens of thousands to approximately 384, and comparing qPCR results with microarray data, DiaGenic narrowed each test down to a 96-gene format run on Life Technologies Applied Biosystems qPCR microfluidic cards run on an ABI 7900 HT system, Lönneborg said.
Both ADtect and BCtect have received the CE Mark for in vitro diagnostics in the EU, based on a study of more than 420 patient samples across eight clinical sites in Europe for ADtect and 550 samples across five clinical sites in the EU and US for BCtect. Both tests — coincidentally, Lönneborg told PCR Insider — demonstrated 73 percent accuracy in detecting disease from blood samples.
"We weren't happy with that at first," Lönneborg said. "But then we wondered how accurate current clinical diagnosis was." In discussions with clinicians, DiaGenic found that the estimated accuracy of current diagnostic methods for Alzheimer's was only around 80 percent.
Further, the company has vetted its tests thoroughly and shown that external factors such as variability in blood collection and testing methods, common co-morbidities, interference from other common medications, contraceptive use, menstrual cycle, and age all had no bearing on the accuracy of either test.
So are clinicians biting? Lönneborg told PCR Insider that although it has sold an undisclosed number of tests in Europe, DiaGenic is currently struggling to convince physicians of the merits of ADtect and BCtect. "Many think that it's too good to be true, to have a simple blood test this accurate," Lönneborg said.
As such, DiaGenic has recently made a point of placing the tests with undisclosed "key opinion leaders in the EU and US" who are currently evaluating their efficacy. Lönneborg also said that DiaGenic has had initial contact with the US Food and Drug Administration about its requirements for a diagnostic approval, but that the company has "limited resources" and is for the time being focusing on Europe.
Pavel Neužil, a researcher at the Korea Institute of Science and Technology-Europe in Saarbrüecken, Germany, discussed his lab's efforts to develop and commercialize an ultrafast and ultracheap real-time PCR system for potential field use and point-of-care diagnostics.
Neužil and colleagues originally developed the system in response to the avian influenza scare in Southeast Asia and other remote locations. He said that his team wanted to develop a system that could detect one or two genes of interest alongside control genes in order to make the fast diagnoses necessary in a rapidly changing infectious disease such as avian flu.
The requirements for the chip were that it had to be economical, disposable, simple to operate, as efficient at detection as more expensive commercial systems, have the ability to detect RNA for certain infectious agents, and be portable and battery operated.
The researchers have developed a prototype of such a system. It uses "virtual reaction chambers" of water-based sample encapsulated in oil droplets on a silicon-etched chip covered with a standard microscope cover slip. Each chip contains four or 10 reaction chambers for genes of interest and control genes, and each reaction chamber is heated using miniature electrical elements.
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A typical sample volume is 100 nL of sample encapsulated in one µL of oil, and the sample can be heated from room temperature to 150 degrees C in as little as 0.2 seconds, allowing the device to perform single-step RT-PCR with 40 thermal cycles in 20 to 30 minutes using three-step thermal cycling.
The chip is placed on a portable optical detection device about the size of a common electronic laboratory scale. The optical detection module is located underneath the chip and is very similar to the optical detection system of a DVD player, Neužil said, using a blue LED excitation source, FITC filter set, and photodiode with amplifier.
The first generation of their device was available in 2007, but since that time, Neužil and colleagues have developed second and third generations of the technology. Key improvements have included simultaneous dual color detection or simplified detection for single colors; increased signal detection sensitivity; a USB power source; and engineering improvements that bring the total expected manufacturing cost to around €100 ($122), Neužil said.
"We believe this is a low enough cost to be able to distribute this widely," Neužil said. "There are a lot of great instruments out there [for qPCR], but they cost too much."
Neužil also said that besides field testing and point-of-care diagnostics, the device will be affordable enough to be used for educational purposes in high schools and universities.
One area that may still need improvement is sample prep, which currently is not integrated onto the platform. Neužil said that they have used magnetic bead-based sample prep to this point, but have not yet determined whether it will be necessary to incorporate it.
Nevertheless, Neužil said that he expects the team will have the latest generation of the device ready for beta testing in three to four months, and that the group is seeking collaborators to commercialize the platform.
High-Res Melting Startup
Researchers at the University of Aarhus in Denmark and the Peter MacCallum Cancer Centre in Melbourne, Australia, have founded a startup company to commercialize a method developed at the institutions to measure post-PCR, locus-specific gene methylation for potential use in in vitro diagnostic applications.
Tomas Wojdacz of the University of Aarhus disclosed the startup, called Epilabs, in his presentation on using high-resolution melting to sensitively and accurately measure single-locus methylation levels.
The technique, called methylation-sensitive high-resolution melting, or MS-HRM, is an adaptation of HRM technology first described by Wojdacz and Alexander Dobrovic of the MacCallum Cancer Centre. It involves PCR amplification of a bisulfite-modified DNA template and subsequent "in tube" HRM analysis of the amplified product.
The bisulfite modification enables the distinction of methylated and unmethylated PCR product on the basis of their different melting temperatures using HRM analysis. In addition, the researchers have designed a proprietary primer that can ensure unbiased co-amplification of methylated and unmethylated templates, resulting in a methylation detection sensitivity in the range of 1 to 0.1 percent, Wojdacz said.
Epilabs has applied for a patent on the technology and is currently exploring commercialization routes.
Helixis PCR Platform
US company Helixis' new miniature real-time qPCR system, called Pixo, is now available for purchase in Sweden and other Nordic countries via Techtum Lab, a Swedish distributor of life science research tools signed on to distribute the platform.
Håkan Evefors, director of sales for Techtum, was demonstrating Pixo at the Techtum conference booth. Evefors said that Helixis' instrument is significant because it costs around €10,000 ($12,200), but retains the performance standards of many other established systems that cost in the neighborhood of €25,000 to €30,000.
Pixo has a bench-top footprint of a little over one square foot and accepts a 48-well reaction plate optimized for 20-µL PCR reactions. The instrument can complete real-time PCR reactions in about 40 minutes using 40 cycles; and its optical system is capable of four-target multiplexing, with factory calibration for SYBR Green I, FAM, HEX, ROX, and Cy5 dyes.
Evefors said that in the US, Pixo will be available from Helixis exclusively.