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German Group Develops On-Chip RPA Arrays for Isothermal Detection of Viruses, Bacteria

Researchers in Germany have developed the first flow-through microfluidic microarray with automated detection using recombinase polymerase amplification (RPA) for on-chip amplification.

The isothermal method was able to detect and quantify a panel of three viruses and bacteria, but researchers said the array size might be virtually unlimited.

In a proof-of-principle study published earlier this month in Analytical Chemistry, researchers at Technical University of Munich, Georg-August-University Göttingen, and Brandenburg Medical School Theodor Fontane, created a water quality assay to detect DNA of Enterococcus faecalis, human adenovirus 41, and Phi X 174.

"If you have a PCR reaction where you know which gene sequence is interesting, then you can very easily adapt your primer for RPA," Michael Seidel, chair of analytical chemistry and of the institute of hydrochemistry at the Technical University of Munich, and a lead researcher on the work, told GenomeWeb in an interview.

"You can design ... and develop any kind of assay in a very short time," he said, noting that this is "a very simple and easy method."

The researchers employed established methods to create the arrays, using Scienion's sciFlexarrayer liquid handling technology to immobilize 500 micron dots of DNA on prepared glass slides.

In the RPA method, a complex made up of recombinase and primer scans the template, invading the double-stranded DNA at homologous sequences. From there, the displaced strand is bound by single-strand DNA-binding proteins, and primers are extended by a polymerase.

The group used biotinylated readouts for the water quality assay, and made the assay quantitative by calibrating it to dilutions of each organism. The total time to run the assay was 48 minutes, according to the study.

The RPA chemistry was originally developed by TwistDx, an Alere subsidiary. It is marketed as a kit for research use, but customers' creative uses of the technology may also inform internal R&D at TwistDx, according to CEO Niall Armes.

"We believe that continuing to supply RPA for research use will lead to developments like this," said Armes.

Seidel's team had previously created a microfluidic system, named MCR 3, which is being commercialized by GWK, a German firm specializing in heating and cooling instruments.

"We have an open-analysis platform, and so the idea is that researchers or industry can use the instrument and have their own application on it," Seidel said.

Over the past 10 years, the group has shown the platform can be used to process "any kind of immunoassay," for example, such as a six-minute chip-based assay to detect 13 different antibiotics in raw milk samples that the group developed.

"We normally use [MCR 3] for immunoassays, but now we have the chance to go to multiplex DNA amplification tests on our system," Seidel said.

Based on the current research, the group is developing a larger project in Germany to create "a fully automated hygiene monitoring system" for drinking water.

"The idea is, we have a system for the concentration of viruses and bacteria in water and our MCR 3 at the end, then you get a signal if there is any contamination," he explained.

The global environmental water quality monitoring market is projected to grow to $20.5 billion by 2020, according to one recent marketing report, due in part to environment-friendly policy changes and expansion of testing in emerging markets.

Meanwhile, the Analytical Chemistry study is "another good example of the promiscuity of the RPA method, showing its capacity to interface with a variety of possible consumables and methods, and to do so 'out-of-the-box'," TwistDx's Armes said.

In particular, the study draws attention to aspects of RPA that the firm sees in its own work as well as that of customers and collaborators.

Namely, the use of RPA on surfaces or in microfluidic devices is "made easy by the capacity of RPA to tolerate off-temperature environments and function on solid phase," Armes said.

Because RPA operates at typical body temperatures — and can even be run using body heat alone — the chemistry enables the use of biological reagents that might not tolerate the heat treatments usually needed for nucleic acid amplification.

"In this case, the authors use biotin-labeled primers and subsequent binding of the enzymes, but one could also readily conceive of how enzymes like [alkaline phosphatase] or [horseradish peroxidase] might be located directly on the oligonucleotides, or how reagents like antibodies might be employed directly," Armes said.

Off-the-shelf, RPA has been employed by a number of research projects and it is also purportedly the chemistry being used in Alere's second-generation influenza assay, as previously reported by GenomeWeb.

Armes said he believes the chemistry is the only rapid isothermal method to offer a broadly applicable alternative to PCR. The firm also recently showed that, in most cases, standard short PCR primers work well in RPA.

"The potential is so large that it cannot conceivably be exploited fully through innovations originating within TwistDx and Alere," he said. "It is, in my view, not only of commercial value but even our duty to make scientists and developers aware of this technology and hope that they will take the time to try it."

For his part, Seidel said that he is open to collaborators to develop more assays for the MCR 3 microfluidic platform. "We are a research institution, but we are interested in this principle getting into the market in the future," he said.

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