Bioluminescence enables rapid DNA detection in infection diagnostics

Imagine: a test with the accuracy of PCR, but faster and simpler. Researchers from Eindhoven University of Technology have developed a new method for the detection of DNA/RNA. PhD student Yosta de Stigter talks about the operation and application of this groundbreaking technology.

 By: Eline te Velde

Polymerase Chain Reaction (PCR) is a reliable and widely used method for detecting bacteria and viruses. Through several cycles of heating and cooling, the DNA multiplies and tiny amounts of viral DNA are made detectable. PCR requires specialist equipment and must be carried out in the laboratory. In practice, this means that the patient has to wait one or more days for a test result. TU Eindhoven and Fontys University of Applied Sciences are working together on an alternative in which the GP makes the diagnosis himself and the patient receives the results within half an hour.

Yosta is investigating the possibility of using this new method to test for STDs. “There is a taboo on the subject. Many people find it difficult to go to the GGD or the GP and ask for a test. It sometimes takes two days until you receive the test results, and during that time you cannot yet start a treatment plan. We hope to accelerate this process and lower the barrier to testing.”

But the researchers mainly see opportunities in countries where central laboratories are not always available. “Infectious diseases, such as malaria, are still a major problem. Simple and cheap tests can really make a big difference.”

LUNAS

Researchers from Eindhoven University of Technology have developed a Luminescent Nucleic Acid Sensor (LUNAS). LUNAS uses so-called luciferase enzymes. These enzymes cause bioluminescence and thus emit light, such as in fireflies and glowworms. Yosta explains how it works: “The sensor detects the DNA of viruses or bacteria in a sample. To find the right DNA we use the D-Cas9 protein.” These proteins are designed to detect and bind to a very specific piece of DNA, for example from a virus.

“The luciferase enzymes help us detect the DNA,” Yosta continues. “We split this into two pieces and attach it to two D-Cas9 proteins. The proteins look for the target DNA and bind to it. When this happens, the two pieces of luciferase enzyme come together and emit light. If the target DNA is not present, the D-Cas-9 proteins do not find each other and it remains dark.” The scam is then made visible with a digital camera.

Amplification

Finding a specific piece of DNA in a microliter sample is highly specialized work. “You can compare it to looking for one fish in Lake Victoria in Africa,” says Yosta. “PCR is so sensitive because it detects a tiny amount of DNA in a sample by amplifying it. The power of DNA is that we can amplify it, which is why DNA tests are the current standard in diagnostics.”

Yosta explains that the LUNAS method itself is not sensitive enough to detect clinically relevant samples, and is therefore combined with an amplification technique. The difference with PCR is that the researchers use an amplification technique that works at a single constant temperature. “We therefore only need two components, something that heats and a camera that can capture the light. This makes the test a lot easier and faster than a PCR test. The combination of speed and a simplified reading method means that in theory you can do this type of test at your GP.”

“The combination of speed and a simplified reading method means that in theory you can do this type of test at your GP.”

From theory to practice

In theory, the LUNAS test can easily be performed at the GP, but to put this into practice, Yosta hopes to integrate the test into a microfluidics device. “We now work with a large box with a camera in it, but that is of course not a setup that the GP can work with. Moreover, it still requires a relatively large amount of laboratory work. We want to make the test more user-friendly.”

According to Yosta, it will certainly take a few more years before the first general practitioners can start using the LUNAS test. “I don't know whether all general practitioners will be using exactly this test in ten years' time, but I dare say that test results will become faster and diagnostics will change in the coming years.”

 Do you want to know more? During the one-day conference “Innovations in DNA & RNA Technologies” on April 16, Yosta de Stigter will give a lecture about the LUNAS method and how it can be applied in a point-of-care environment. Visit the website for more information and Sign In for a free visit.