With the number of COVID-19 cases increasing again worldwide and no effective treatment in sight, the need for frequent and large-scale testing has become even more important. The current gold-standard uses a technique called “qRT-PCR” (quantitative real-time polymerase chain reaction), which is highly sensitive and specific. However, as well as needing a turnaround time of several hours, it must be performed in a lab since it requires specialized equipment. So not only is qRT-PCR out of the reach of anyone at home, it is even too costly for many labs around the world.
Now, in a collaborative effort, PhD students at the Research Institute of Molecular Pathology (IMP) and Institute of Molecular Biotechnology (IMBA) in Vienna have developed an alternative strategy that overcomes several of the previous limitations. Under the guidance of Julius Brennecke and Andrea Pauli, students Max Kellner, Julian Ross and Jakob Schnabl have teamed up to produce this innovative method.
A Series of Clever Changes
The principle behind COVID-19 testing is to isolate and amplify the small pieces of genetic material called RNA, the ancient cousin of DNA, that the SARS-CoV-2 virus brings into our cells when we are infected. The problem is that it’s often tricky to obtain the quantity and quality needed for testing since RNA quickly degrades outside of cells. Isolating enough RNA normally requires careful handling of samples with hazardous chemicals in a lab.
What the VBC team has shown is that heating saliva samples for five minutes with an easy-to-handle commercial reagent, followed by “fishing out” the RNA with magnetic beads, can extract more than enough RNA for testing.
To simplify the test further, the scientists also improved the technique used to amplify the genetic material of the virus. The current method, qRT-PCR, cycles samples through different temperatures at thirty second intervals for over an hour – almost impossible to achieve without a specially-dedicated machine. Now, this has been replaced by “RT-LAMP” (loop-mediated isothermal amplification), which requires only that the sample be held at a steady 63°C for 30 minutes. This has proven equally sensitive and robust in detecting SARS-CoV-2.
Finally, the scientists introduced a simple colour change reaction to show the results. At the end of the 30 minutes, a SARS-CoV-2 positive sample will change from violet to sky blue. This is much simpler than in conventional tests measuring levels of a particular fluorescent molecule, which is detectable only with lab equipment.
By “tweaking at different places where we saw room for improvement,” the team reports, they have developed a testing strategy that performs as well as the method currently used in hospitals, in just a fraction of the time.
No Lab Equipment Needed
One of the team’s goals was a test that could be completely carried out at home, requiring only plastic tubes and strips of filter paper. Dubbing the procedure “HomeDip-LAMP”, the procedure simply calls for the user to mix his or her saliva with several provided reagents, and to transfer the mixtures between tubes using filter paper. The hardest step is to maintain the 63°C needed for the 30-minute incubation period, but the scientists have shown that this too can be done using a sous-vide cooker, or by mixing boiling and room temperature water.
In an interesting twist to the many plans thwarted by lockdown, this project came about only because Austrian Max Kellner was stranded at home in Vienna by travel restrictions. Kellner, a PhD student at the LMB institute in Cambridge, joined with fellow students Julian Ross and Jakob Schnabl at the VBC to see what they as scientists could do to help in the pandemic.
“We felt like we just had to do something,” said Ross, who asked to join the efforts midway into the lockdown – in keeping with the “collaborative spirit” the students described among the 1,300 scientists from 80 countries engaged in research at the VBC.
Indeed, the development of HomeDip-LAMP could now transform the ability of the population to quickly and reliably test themselves without even leaving their homes. The study will now be reviewed by fellow scientists before possibly undergoing clinical certification. “We would be very happy to see it move into a kit or testing strategy that countries adopt for decentralized testing,” Schnabl told Metropole.
Find out more about this study as a preprint on bioRxiv.