A fast new test that fishes for multiple respiratory viruses at once using single strands of DNA as bait has been developed by researchers at the University of Cambridge.

The test, which gives highly accurate results in just one hour, uses DNA “nanobait” and nanopore sensing technology to detect short RNA targets from the most common respiratory viruses—including influenza, rhinovirus, respiratory syncytial virus (RSV), and COVID-19—at the same time. The approach requires no amplification steps and enables near 100% specificity due to the precision of the programmable nanobait structures. In comparison, PCR tests, while highly specific and highly accurate, can only test for a single virus at a time and take several hours to return a result.

The technology can be used in any setting, and in a newly reported study the team showed how the test can be easily modified to detect different bacteria and viruses, and viral variants. In a newly reported study, the researchers described the evaluation of the technology on swab samples from SARS-CoV-2 patients. The researchers said that by testing for multiple viruses at once, their test will help to ensure that patients get the right treatment quickly, and could also reduce the unwarranted use of antibiotics.

“This work elegantly uses new technology to solve multiple current limitations in one go,” said Stephen Baker, PhD, from the Cambridge Institute of Therapeutic Immunology and Infectious Disease. “One of the things we struggle with most is the rapid and accurate identification of the organisms causing the infection. This technology is a potential game-changer; a rapid, low-cost diagnostic platform that is simple and can be used anywhere on any sample.”

Baker is a co-author of the team’s published study in Nature Nanotechnology, which is headed by first author Filip Bošković from Cambridge’s Cavendish Laboratory, and titled “Simultaneous identification of viruses and viral variants with programmable DNA nanobait,” in which they wrote, “Our system allows for multiplexed identification of native RNA molecules, providing a new scalable approach for diagnostics of multiple respiratory viruses in a single assay.”

Diagnosing infectious diseases is important for determining the most appropriate form of treatment, the authors noted. But while respiratory tract infections are a major cause of infectious disease death globally, “many respiratory viruses induce comparable symptoms and cannot be differentiated clinically, making the identification of appropriate treatment challenging.”

“Many respiratory viruses have similar symptoms but require different treatments: we wanted to see if we could search for multiple viruses in parallel,” said Bošković, who is a PhD student at St John’s College, Cambridge. “According to the World Health Organization, respiratory viruses are the cause of death for 20% of children who die under the age of five. If you could come up with a test that could detect multiple viruses quickly and accurately, it could make a huge difference.”

Currently, viral diagnostics rely on quantitative reverse transcription-PCR (qRT-PCR), followed by genome sequencing to detect viral variants, the authors explained. PCR-based diagnostic methods provide a sensitive approach for detecting viral nucleic acids in complex biological samples, but there’s limited ability for multiplexing. “There is a need for robust diagnostic methods that can simultaneously detect multiple respiratory viruses and variants in limited sample volume, which can be quickly reconfigured to detect additional variants as they arise.” And while PCR tests are powerful, sensitive, and accurate, the team continued, they require a piece of genome to be copied millions of times, which takes several hours.

“Good diagnostics are the key to good treatments,” said Bošković. “People show up at the hospital in need of treatment and they might be carrying multiple different viruses, but unless you can discriminate between different viruses, there is a risk that patients could receive incorrect treatment.”

The Cambridge researchers wanted to develop an alternative type of test that uses RNA to detect viruses directly, without the need to copy the genome, but with high enough sensitivity to be useful in a healthcare setting.

“For patients, we know that rapid diagnosis improves their outcome, so being able to detect the infectious agent quickly could save their life,” said Baker. “For healthcare workers, such a test could be used anywhere, in the U.K. or in any low- or middle-income setting, which helps ensure patients get the correct treatment quickly and reduce the use of unwarranted antibiotics.”

The researchers based their new test on structures built from double strands of DNA with overhanging single strands. These single strands are the “bait,” and are programmed to “fish” for specific regions in the RNA of target viruses. The nanobaits are then passed through the tiny nanopores. Nanopore sensing is like a ticker tape reader that transforms molecular structures into digital information in milliseconds. The structure of each nanobait reveals the target virus or its variant. “We employed programmable viral RNA cutting with RNase H to remove short RNA targets that uniquely identify the virus,” the investigators explained. “The resultant RNA target is captured by nanobait, which is detected immediately by nanopore sensing, without reverse transcription, pre-amplification, or purification.”

In their reported study, the team showed that the platform could concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. “Our nanobait can be easily reprogrammed to discriminate viral variants, as we demonstrated for several key SARS-CoV-2 variants with single-nucleotide resolution,” the scientists added. They also demonstrated how the nanobait approach could discriminate between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients without pre-amplification.

The authors concluded, “As nanobait has proven to be specific and accurate for viral detection in patient samples, we think our platform can be employed for native RNA detection. Nanobait paves a way for a multiplexed amplification-free RNA detection method that is dependent only on the rapid single-molecule readout of the nanobait structure.”

“Bringing together researchers from medicine, physics, engineering, and chemistry helped us come up with a truly meaningful solution to a difficult problem,” said Bošković, who received a 2022 PhD award from the Cambridge Society for Applied Research for this work.

A patent on the new technology has been filed by Cambridge Enterprise, the University’s commercialization arm, and co-author Ulrich Keyser, PhD, has co-founded a company, Cambridge Nucleomics, focused on RNA detection with single-molecule precision.

“Nanobait is based on DNA nanotechnology and will allow for many more exciting applications in the future,” said Keyser, who is based at the Cavendish Laboratory. “For commercial applications and roll-out to the public we will have to convert our nanopore platform into a hand-held device.”