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Chinese Researchers Create Paper-Based, Colorimetric Test to Detect SARS-CoV-2 Variants


NEW YORK – As new SARS-CoV-2 variants and subvariants keep appearing, creating new challenges for testing and trying to manage the COVID-19 pandemic, a team of researchers from multiple universities in China has developed a rapid paper-based test to detect mutations to the virus. 

The team, which includes academics from Tsinghua University in Beijing, Zhengzhou University in Zhengzhou, and Sichuan University in Chengdu, laid out the development of its low-cost assay in a paper published in Nature Biomedical Engineering last week.

The underlying principle of the test dubbed MARVE relies on a double-stranded DNA probe designed to recognize the mutation markers that indicate a specific variant, according to Ruijie Deng, one of the authors of the paper and an associate professor in the college of biomass science and engineering at Sichuan University.

When RNA from the virus that contains one of those markers is present in a sample, the interaction between the RNA and the DNA probe, combined with an enzyme, releases silver and produces ammonium, which changes the pH of the entire solution. The change in pH is measured by phenol red, a pH indicator that is red when the pH of a solution is below seven but turns yellow when the pH increases to eight, Deng said.

All of the tools for the test, besides a heat block and reagents to quickly extract the viral RNA, are included on a piece of origami paper. One section contains the sample's nucleic acids, one has the probes, one has a urease enzyme, and one has urea and phenol red. The sample section folds to apply the sample to the probes and is incubated for 20 minutes to release the silver. The combined section is folded again to apply the urease enzyme, then folded a final time to apply the urea and phenol red. Combining urease and urea forms ammonium, which results in the pH change that is picked up by phenol red and indicates the result.

Deng said the researchers designed the DNA probes to be stable and a perfect match with a specific variant, so only the presence of that variant can cause the reaction. The test can be designed to detect any number of variants, as multiple detection sites with the variant-specific probes can be printed onto one piece of paper. The user would know which variant is present in the sample depending on which section of the paper changes color.

While a user can detect the color change using the naked eye, the team developed a phone app that uses the cellphone camera to ensure a more precise result. A user can take a picture of the result and upload it to the app, where an analyzer processes the image's color to determine the variant. Including the working time of the app and the RNA extraction step, the process takes about 30 minutes, Deng said. It's a little longer than a lateral flow antigen test, but significantly shorter than sequencing, and "very cheap" compared to PCR — it costs about $.30 for a test that covers the N and E gene of SARS-CoV-2 and five variants.

In the Nature paper, the team tested 50 throat swab samples and found the assay detected the presence of SARS-CoV-2, along with mutations specific to the Alpha, Beta, and Gamma variants, with 100 percent concordance with PCR and sequencing. Although the team didn't include Omicron in the paper, because it was not as widespread when submitting the data, the test works with the variant and "will be workable" for subvariants of Omicron, Deng said.

The test may also be able to detect new variants. Because it "can be programmable to detect multiple mutations," he said, "if we design probes to detect genome sites that are likely to be mutated," new variants could be discovered. That ability is "dependent on the multiplexing capacity of the assay, and its improvement can be achieved by printing more detection sites."

Even though the researchers have tested some clinical samples, more clinical validation is needed before the assay could be launched commercially, he said, but that validation is being done. A commercial launch would likely require collaboration with a diagnostics company, Deng said.

He noted that the test might be "very promising" for use at home since "the operation … is very simple." The biggest advantage is the ability to delineate between different variants, which could help governments or public health organizations monitor outbreaks before they spread. The tests could be used to "investigate which and how frequently the SARS-CoV-2 variants spread," as well as the distribution of the variants across a population. 

Because different variants "show a different capacity to infect" people, having a test that determines which variant is present in a sample can allow for "more precise interventions for controlling the spread" and "more efficient ways" to address infections and incidence rates, he added.

Although the test has clear potential uses in low- and middle-income countries as well, the researchers don't have the resources to distribute it but are open to working with companies that have the interest and experience to bring the test to that market, Deng said.