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Simpler Method Devised to Validate Covid-19 Tests

Covid-19 testing


2 Dec. 2020. An engineering lab created simple and stable control probes to validate widely used diagnostic tests for SARS-Cov-2 viruses responsible for Covid-19. The control techniques, acting as accuracy benchmarks, are described by researchers from University of California in San Diego, in the 25 Nov. issue of the journal ACS Nano (paid subscription required).

The UC-San Diego team from the lab of Nicole Steinmetz, professor of nanoengineering, is seeking simple and reliable methods for validating the RT-PCR test, considered the gold standard for detecting the SARS-CoV-2 virus in patients. RT-PCR stands for reverse transcription – polymerase chain reaction, a type of genetic analysis looking for the viral RNA signature indicating SARS-CoV-2 in respiratory fluid samples.

RT-PCR tests, however, need validation that use positive controls, true-north benchmarks that assure the test is accurately measuring or detecting its target, in this case the presence of SARS-CoV-2 viral RNA. Today’s RT-PCR validation techniques, say the authors, use RNA samples from infected patients, synthetic RNA, or circular strands of DNA called plasmids. These RNA or plasmid samples degrade easily and require refrigeration, making them difficult to use outside of lab or clinical settings.

“Our goal,” says Steinmetz in a university statement, “is to make an impact not necessarily in the hospital, where you have state-of-the-art facilities, but in low-resource, underserved areas that may not have the sophisticated infrastructure or trained personnel.”

Steinmetz’s nano-engineering lab studies viruses at the nanoscale level, particularly viruses from plants. The lab maintains a library of plant viruses and nanoscale particles from these viruses that act as biotech building blocks for biomedical and agricultural applications. For validating SARS-CoV-2 detection tests, the team led by postdoctoral researcher and first author Soo Khim Chan designed two virus-like nanoparticles that mimic RNA from SARS-CoV-2 viruses and perform the same positive control functions as current methods, but are more stable and easier to use.

The researchers devised one virus-like nanoparticle from a plant virus, the cowpea chlorotic mottle. This virus infects cowpeas, also known as black-eyed peas, but also provides a framework for building synthetic virus-like particles. In this case, the team filled out the viral framework with RNA sequences from the SARS-CoV-2 virus.

The second viral nanoparticle is built around viruses called bacteriophages, natural enemies of bacteria that infect and replicate inside the microbes. The team inserted plasmids with genes coding for SARS-CoV-2 viruses into a bacteriophage framework and coated the matrix with surface proteins from Qbeta bacteriophages. When bacteria ingest the synthetic bacteriophage, they produce virus-like particles with SARS-CoV-2 sequences, coated with Qbeta bacteriophage proteins.

Tests of both virus-like nanoparticles with clinical samples show they work as well as conventional positive control methods for RT-PCR tests, and can also validate the entire testing process, which the authors say cannot be done with today’s methods.

“We can use these as full process controls,” says Chan referring to the virus-like nanoparticles. “We can run the analysis in parallel with the patient sample starting all the way from RNA extraction. 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.”

Another key advantage is the virus-like nanoparticles’ stability. The authors say the nanoparticles can be stored at 40 degrees C (104 F) for a week, and remain 70 percent active, i.e. resemble SARS-CoV-2 viruses, after a month. While the paper reports on validation of RT-PCR tests, the authors say their validation techniques can be adapted to reverse-transcription loop-mediated isothermal amplification, or RT-LAMP tests used in point-of-care or at-home tests.

“It’s a relatively simple nanotechnology approach to make low-tech assays more accurate,” notes Steinmetz. “This could help break down some of the barriers to mass testing of underserved populations in the U.S. and across the world.”

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