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Nanoengineers from the University of California at San Diego have developed new and improved probes, known as positive controls, that could make it easier to validate rapid and point-of-care diagnostic tests for COVID-19 around the world.
Positive controls, consisting of virus-like particles, are stable and easy to produce. The 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 laboratory.
“Our goal is to make an impact not necessarily in the hospital, where you have state-of-the-art facilities, but in areas with few resources and under-served that may not have sophisticated infrastructure or qualified personnel,” said Nicole Steinmetz. a professor of nanoengineering at UC San Diego Jacobs School of Engineering.
Positive controls are a fundamental element in the laboratory: they are used to verify that a test or experiment really works. The positive controls used primarily to validate today’s COVID-19 tests are naked synthetic RNAs, plasmids, or RNA samples from infected patients. But the problem is RNA and plasmids are not as stable as viral particles. They can degrade easily and require refrigeration, making them inconvenient and expensive to ship around the world or to store for long periods of time.
In an article published on 25 November a ACS Nano, UC San Diego researchers led by Steinmetz report that by packaging RNA segments 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 maintain 70% of their activity even after one month of storage and can pass detection as a new coronavirus without being contagious.
The team developed two different controls: one consisting of plant virus nanoparticles, the other of bacteriophage nanoparticles. Using them is simple. Controls are run and analyzed along with a patient sample, providing a reliable reference point for what a positive test result should look like.
To carry out the virus-based controls of the plants, the researchers use the cowpea chlorotic patch virus, which infects black-eyed pea plants. They essentially open the virus, remove its RNA content, replace it with a synthesized RNA template containing specific sequences from the SARS-CoV-2 virus, then close everything again.
The process of making bacteriophage-based controls begins with plasmids, which are rings of DNA. Inserted into these plasmids are the gene sequences of interest of the SARS-CoV-2 virus, as well as the genes that encode the surface proteins of the bacteriophage Qbeta. These plasmids are then absorbed by the 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 specimens. A big advantage, the researchers point out, is that unlike the positive controls used today, these can be used at all stages of a COVID-19 test.
“We can use them as complete process controls – we can run the analysis in parallel with the patient sample starting with the RNA extraction,” said first author Soo Khim Chan, a postdoctoral researcher in Steinmetz’s lab. “Usually other controls are added in a later step. So if something went wrong in the first few steps, you won’t be able to know.”
So far, researchers have adapted their controls for use in the CDC-authorized RT-PCR assay. 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 the samples to a PCR-capable lab.
Steinmetz, Chan and colleagues are now working on adapting the controls for use in less complex diagnostic tests such as the RT-LAMP test which can be run on-site, out of the lab and deliver results immediately.
“This is a relatively simple nanotechnology approach to making low-tech testing more accurate,” Steinmetz said. “This could help break down some of the barriers to mass testing of disadvantaged populations in the United States and around the world.”
This work was funded in part by grants from the National Science Foundation: RAPID CBET-2032196 and RAPID CMMI-2027668, as well as the University of California: UCOP-R00RG2471 and a Galvanizing Engineering in Medicine (GEM) award.
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