Researchers based at the University of Minnesota Medical School have developed two tests using CRISPR-Cas9 gene editing technology that can quickly and accurately diagnose SARS-CoV-2 infection.
The first test is a lateral flow antigen test designed for rapid, at home use by non-experts and is designed to differentiate between COVID-19 variants. It can produce results within an hour and works in a similar way to a pregnancy test.
The second test uses fluorescence and is more sensitive and more complex. It can differentiate between SARS-CoV-2, influenza A and B, and respiratory syncytial virus. Although this test needs to be done in a laboratory, it can also be completed within an hour, does not require overly specialized equipment and has the potential to be scaled up for large scale testing.
“The approval of the SARS-CoV-2 vaccine is highly promising, but the time between first doses and population immunity may be months,” said Mark Osborn, Ph.D., assistant professor at the Medical School and first author of the paper describing the research, which is published in the journal Bioengineering. “This testing platform can help bridge the gap between immunization and immunity.”
The team used CRISPR-Cas9 to target the ORF8a section of the SARS-CoV-2 genome — the CDC and WHO currently test for sequences in the N and E genes of the virus. The researchers chose this target, because it differs significantly between different coronaviruses.
The lateral flow test is designed for use in the field and uses gold nanoparticles coated with rabbit antibodies. In order to generate a positive result both fluorescein isothiocyanate (FITC) and biotin have to be bound on the test strip. If only FITC is bound then the test is active, but not positive.
The second test is a fluorescence-based assay that can be run on a RT-PCR machine that can detect the ORF8a sequence of SARS-CoV-2. This test uses a DNA probe with a fluorophore attached that can bind the target after a 20-minute incubation period during which it is cut by Cas9 causing it to light up.
When the researchers added three additional probes to detect influenza A and B and respiratory syncytial virus there was no significant cross reactivity between the probes and it was possibly to clearly differentiate between the four viruses in a given sample.
The fluorescence test had similar accuracy to RT-PCR and had a cycle threshold or CT level of 34.9. The CT value is used to show how much virus someone infected with SARS-CoV-2 or other viruses has in their system. CDC guidelines say the CT level of a test should be less than 40 for a positive test.
Sensitivity of the lateral flow assay test was less good, although comparable to other similar tests being used in the field. This lower detection level “may result in an inability to detect patients with low viral titers and makes follow-on confirmation of rapid tests important,” write the authors.
“Our approach adds to the armamentarium of testing methodologies that can be brought to bear to bridge the immunization–immunity gap,” concludes the team, who are now seeking to further test, commercialize and scale up these diagnostics.
