Rapid COVID-19 tests are on the rise to provide faster results to more people, and scientists need an easy and foolproof way to know that these tests are working properly and the results can be trusted. Nanoparticles undergoing discovery, such as the new Coronavirus, may be just the ticket.
Such a coronavirus-like nanoparticle, developed by nanoscale engineers at UCSD, will serve as something called positive control in COVID-19 tests. Positive controls are samples that are always positive. They are run and analyzed alongside patient samples to verify that tests for COVID-19 are running consistently and as intended.
Positive controls developed at UCSD offer several advantages over those currently used in testing for COVID-19: They do not need to be kept cold; It is easy to manufacture. They can be included in the entire testing process from start to finish, just like a patient sample; And because they are not actual virus samples taken from COVID-19 patients, they do not pose a risk of infection to the people who take the tests.
The researchers, led by Nicole Steinmetz, a professor of nanoscale engineering at the University of California, San Diego, have published their work in the journal. Biomolecules.
This work builds on an earlier version of the positive controls developed by Steinmetz’s Laboratory for RT-PCR testing, which is the gold standard for testing for COVID-19. Positive controls could be used in the new study 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 instantly and provide results in about an hour.
Steinmetz said having a robust tool to ensure these tests are run accurately – especially for low-tech diagnostic assays like RT-LAMP – is critical. It can help enable rapid comprehensive testing for COVID-19 in under-resourced, disadvantaged areas and other places that do not have access to advanced testing equipment, specialized reagents, and trained professionals.
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The new positive controls are essentially small viral sheaths – made from either a plant virus or phage – that contain bits of coronavirus RNA inside. RNA segments include binding sites for both the prefixes used in the PCR and LAMP assays.
“This design creates an all-in-one control that can be used for any of these tests, which makes it very versatile,” said first author Su Khem Chan, a postdoctoral researcher at Steinmetz’s lab.
The team developed two types of positive controls. One of them is made of plant virus nanoparticles. To manufacture them, researchers infected cowpea plants in the laboratory with the green cowpea virus, then extracted the viruses from the plants. Next, the researchers removed the virus’s RNA and replaced it with a specially designed RNA model that contained specific but non-infectious sequences of SARS-CoV-2. The resulting nanoparticles are made from the Coronavirus RNA sequence packaged inside plant virus coatings.
Other positive control was performed by phage nanoparticles. It implies a similar recipe. The researchers infected the E.coli bacteria with custom-made plasmids – loops of DNA – containing specific portions of the (which are also non-infectious) sequences of the SARS-CoV-2 virus, as well as genes encoding for surface proteins of a. Bacteriophages called Qbeta. This process caused the bacteria to produce nanoparticles that consist of the Coronavirus RNA sequence packed inside the bacteria’s shells.
Plant virus and phage shells are key factors making these positive controls so strong. They protect coronavirus RNA slices from breaking down in warmer temperatures – tests have shown they can be stored for a week at temperatures up to 40 ° C (104 ° F). The shells also protect RNA during the first step of PCR and LAMP tests, which involves breaking down cells in the sample – via enzymes or heat – to free their genetic material for testing.
There are no such protections in the positive controls currently used in testing for COVID-19 (naked synthetic RNA samples, plasmids, or RNA from infected patients). This is why current controls either require cooling (making them uncomfortable to handle, and expensive to ship and store) or they must be added at a later stage of testing (which means scientists won’t know if something is wrong with the first steps) .
As a next step, the researchers are looking to partner with industry to implement this technology. Steinmetz’s team said that the positive controls could be adapted to any RT-PCR or RT-LAMP assay, and that using them would help nullify the false readings. Additionally, these positive controls can easily be produced in large quantities by either molecular cultivation in plants or microbial culture fermentation, which is good news to translate into large scale industrialization.
“With mutations and variants emerging, constant testing will be essential to keep the population safe,” said Steinmetz. “The new technology could find a particular benefit for tests at home, which may have a higher rate of false readings due to less controlled experimental conditions.”
Paper title: “Virus-like particles as positive controls for COVID-19 RT-LAMP diagnostic assays.”
This work was funded in part by the National Science Foundation (RAPID CBET-2032196 and RAPID CMMI-2027668) and the University of California (UCOP-R00RG2471 and Galvanizing Engineering Award in Medicine).