COVID-19 rapid tests are on the rise to deliver results faster to more people, and scientists need a simple, foolproof way to know these tests are working properly and that the results can be trusted. Nanoparticles that pass detection as a novel coronavirus might just be the ticket.
Such coronavirus-like nanoparticles, developed by nano-engineers at the University of California at San Diego, would serve as something called a positive control for COVID-19 testing. Positive controls are samples that test consistently positive. They are run and analyzed alongside patient samples to verify that COVID-19 tests are working consistently and as intended.
The positive controls developed at UC San Diego offer several advantages over those currently used in COVID-19 tests: they do not need to be kept cold; they are easy to manufacture; they can be included in the whole testing process from start to finish, just like a patient sample; and since these are not real virus samples from COVID-19 patients, they do not pose a risk of infection to people performing the tests.
Researchers led by Nicole Steinmetz, professor of nanotechnology at UC San Diego, published their work in the journal Biomacromolecules.
This work builds on an earlier version of the positive controls that Steinmetz’s lab developed for the RT-PCR test, which is the gold standard for the COVID-19 test. The positive controls in the new study can be used not only for the RT-PCR test, but also for a cheaper, simpler, and faster test called the RT-LAMP test, which can be performed on-site and provide results in about one o’clock. .
Having a robust tool to ensure these tests are performed accurately – especially for low-tech diagnostic tests like RT-LAMP – is essential, Steinmetz said. This could allow rapid and massive testing of COVID-19 in low-resource and underserved areas and other places that lack access to sophisticated testing equipment, specialized reagents and trained professionals.
Improved positive controls
The new positive controls are essentially tiny virus shells – made up of plant viruses or bacteriophages – that house segments of coronavirus RNA inside. RNA segments include binding sites for both primers used in PCR and LAMP assays.
“This design creates an all-in-one control that can be used for either of these tests, making it very versatile,” said lead author Soo Khim Chan, who is a postdoctoral researcher in the Steinmetz laboratory.
The team developed two types of positive controls. One was made from nanoparticles of plant viruses. To make them, the researchers infected cowpea plants in the lab with cowpea chlorotic mottle virus, then extracted the viruses from the plants. Next, the researchers removed the RNA from the virus and replaced it with a personalized RNA template containing specific but non-infectious sequences of the SARS-CoV-2 virus. The resulting nanoparticles are made up of coronavirus RNA sequences packaged inside plant virus shells.
The other positive control consisted of bacteriophage nanoparticles. It was a similar recipe. Researchers infected E. coli bacteria with custom-made plasmids; DNA rings-; which contain specific fragments of sequences (which are also non-infectious) of the SARS-CoV-2 virus, as well as genes encoding surface proteins of a bacteriophage called Qbeta. This process caused the bacteria to produce nanoparticles made up of coronavirus RNA sequences packaged inside bacteriophage shells.
Plant virus and bacteriophage shells are essential to make these positive controls so robust. They protect the coronavirus RNA segments from degradation at warmer temperatures; tests have shown that they can be stored for a week at temperatures up to 40 ° C (104 ° F). The shells also protect the RNA during the first step of PCR and LAMP testing, which involves breaking down the cells in the sample -; via enzymes or heat -; to release their genetic material for testing.
These protections are not present in the positive controls currently used in COVID-19 tests (naked synthetic RNAs, plasmids or RNA samples from infected patients). This is why existing controls require refrigeration (making them inconvenient to handle, expensive to ship and store) or need to be added at a later stage of testing (meaning scientists won’t know if anything went wrong in the early stages.).
In the next step, researchers seek to partner with industry to implement this technology. Positive controls can be adapted to any established RT-PCR or RT-LAMP assay, and their use would help nullify false readings, the Steinmetz team said. In addition, these positive controls can be easily produced in large quantities by molecular farming in plants or fermentation by microbial culture, which is good news for translating them into large-scale manufacturing.
“With the emergence of mutants and variants, further testing will be imperative to ensure the safety of the population,” said Steinmetz. “The new technology could find utility especially for home testing, which may have a higher rate of false reads due to the less controlled experimental conditions.”
University of California San Diego
Chan, SK, et al. (2021) Virus-like particles as positive controls for COVID-19 RT-LAMP diagnostic tests. Biomacromolecules. doi.org/10.1021/acs.biomac.0c01727.