Illustration and TEM image of
SARS-CoV-2 positive control made from plant virus-based nanoparticles (left)
and bacteriophage nanoparticles (right). Image courtesy of Soo Khim Chan/ACS
Nano
Nanoengineers
at the University of California San Diego have developed new and improved
probes, known as positive controls, that could make it easier to validate
rapid, point-of-care diagnostic tests for COVID-19 across the globe.
The
positive controls, made from virus-like particles, are stable and easy to
manufacture. Researchers say the controls have the potential to improve the
accuracy of new COVID-19 tests that are simpler, faster and cheaper, making it
possible to expand testing outside the lab.
“Our goal
is to make an impact not necessarily in the hospital, where you have
state-of-the-art facilities, but in low-resource, under served areas that may
not have the sophisticated infrastructure or trained personnel,†said Nicole
Steinmetz, a professor of nanoengineering at the UC San Diego Jacobs School of
Engineering.
Positive
controls are a staple in the lab—they are used to verify that a test or
experiment indeed works. The positive controls that are primarily used to
validate today’s COVID-19 tests are naked synthetic RNAs, plasmids or RNA
samples from infected patients. But the issue is RNA and plasmids are not
stable like viral particles. They can degrade easily and require refrigeration,
making them inconvenient and costly to ship around the world or store for long
periods of time.
In a paper
published Nov. 25 in ACS Nano, UC San Diego researchers led by Steinmetz report
that by packaging segments of RNA from the SARS-CoV-2 virus into virus-like
particles, they can create positive controls for COVID-19 tests that are
stable—they can be stored for a week at temperatures up to 40 C (104 F), and
retain 70% of their activity even after one month of storage—and can pass
detection as the novel coronavirus without being infectious.
The team
developed two different controls: one made from plant virus nanoparticles, the
other from bacteriophage nanoparticles. Using them is simple. The controls are
run and analyzed right alongside a patient sample, providing a reliable
benchmark for what a positive test result should look like.
To make the
plant virus-based controls, the researchers use the cowpea chlorotic mottle
virus, which infects black-eyed pea plants. They essentially open the virus,
remove its RNA contents, replace them with a synthesized RNA template
containing specific sequences from the SARS-CoV-2 virus, then close everything
back up.
The
process to make the bacteriophage-based controls starts with plasmids, which
are rings of DNA. Inserted into these plasmids are the gene sequences of
interest from the SARS-CoV-2 virus, as well as genes coding for surface
proteins of the bacteriophage Qbeta. These plasmids are then taken up by
bacteria. This process reprograms the bacteria to produce virus-like particles
with SARS-CoV-2 RNA sequences on the inside and Qbeta bacteriophage proteins on
the outside.
Both
controls were validated with clinical samples. A big advantage, the researchers
point out, is that unlike the positive controls used today, these can be used
in all steps of a COVID-19 test.
“We can
use these as full process controls—we can run the analysis in parallel with the
patient sample starting all the way from RNA extraction,†said first author Soo
Khim Chan, a postdoctoral researcher in Steinmetz’s lab. “Other controls are
usually added at a later step. So if something went wrong in the first steps,
you won’t be able to know.â€
So far,
the researchers have adapted their controls for use in the CDC-authorized
RT-PCR test. While this is currently the gold standard for COVID-19 testing, it
is expensive, complex, and can take days to return results due to the logistics
of sending samples off to a lab with PCR capability.
Steinmetz,
Chan and colleagues are now working on adapting the controls for use in less
complex diagnostic tests like the RT-LAMP test that can be done on the spot, out
of the lab and provide results right away.
“It’s a
relatively simple nanotechnology approach to make low-tech assays more
accurate,†Steinmetz said. “This could help break down some of the barriers to
mass testing of underserved populations in the U.S. and across the world.â€