C.Changes to the SARS-CoV-2 genome – including some found in currently circulating variants – may adversely affect detection of the virus by reverse transcription (RT) PCR, such as one reported on April 26 in Journal of Clinical Microbiology. The researchers suggest that mutations in the loci recognized by DNA primers reduce the amplification of viral sequences and, as a result, may hinder the detection of the virus in samples from COVID-19 positive individuals.
This finding is not a cause for full-blown panic, say the authors. “We thought that might be more common than not. But it turns out it’s actually pretty rare, ”says co-author David Wang, a virologist at Washington University. Wang and colleagues recommend that diagnostic tests span more than one target to ensure proper SARS-CoV-2 detection. While a number of products already contain multiple genetic targets, some emergency-approved COVID-19 RT-PCR assays only check one.
The primers used for RT-PCR assays were developed early in the pandemic when the SARS-CoV-2 virus was first sequenced. Based on information about other coronaviruses, researchers developed PCR primers to amplify sequences in the viral genome that are believed to remain relatively stable. The approach has been used with great success to detect SARS-CoV-2 in samples from nasopharyngeal swabs, saliva and even wastewater.
At Washington University, the Molecular Diagnostic Laboratory at Barnes-Jewish Hospital has used the Roche cobas SARS-CoV-2 test to process patient samples. It looks for the viral gene. ORF1ab as well as the E gene, which encodes the coat protein. For any given sample, these targets should take approximately the same number of PCR cycles to be detected, a value known as the cycle threshold.
“If you have a high value in one gene, you have a high value in the other gene – and vice versa,” says co-author Bijal Parikh, clinical pathologist at Washington University and medical director of the molecular diagnostic laboratory. While most of the samples his team processed had similar cycle thresholds for the two targets, a handful deviate from the expected correlation: sometimes the E gene wasn’t amplified to the same extent as it was ORF1ab.
Despite this strange result, the tests identified SARS-CoV-2 positive samples based on the ORF1ab Signal. To find out what was wrong with the E gene, the team sequenced a handful of virus samples. They found that three samples shared a common mutation in the E gene, one of which was not present in any of the common variants now circulating in the population. The researchers suggest that the mutation affects the binding of the PCR primer and interferes with amplification.
Unfortunately, they were unable to confirm this hypothesis because the primer and probe sequences of the assay are not publicly available. However, the mutated site falls within the sequence targeted by primers used in a widely used E-gene assay developed by researchers at Charité – Universitätsmedizin Berlin in Germany.
Another study published in September in the Journal of Clinical Microbiology reported similar results where mutations elsewhere within the E gene sequence targeted by the Charité primer probe set also interfered with RT-PCR in the Roche cobas SARS-CoV-2 assay. And in December 2020, Emily Crawford, a biochemist at the University of California at San Francisco, and colleagues at the reported Journal of Clinical Microbiology A related finding: Mutations in the SARS-CoV-2 N gene, which codes for the virus’s nucleocapsid protein, reduced the sensitivity of the RT-PCR test by disrupting primer binding.
“We’re still learning which regions of the virus are most conserved and which are the least conserved,” says Crawford. “Until we really hold onto that, we have to be on our toes to be ready for a mutation to pop up somewhere.”
The scientific community needs more such studies, says Chantal Vogels, a virologist at the Yale School of Public Health who was not involved in the new work. She led a study published in July 2020 in Natural microbiology that analyzed the sensitivity and efficiency of SARS-CoV-2 primer sets. The researchers found that a mismatch between a primer and its target sequence resulted in decreased test sensitivity.
The U.S. Food and Drug Administration (FDA) has recognized that SARS-CoV-2 mutations could interfere with COVID-19 testing and continues to monitor variants and evaluate possible effects on diagnostic assays. The agency has also made recommendations to developers to design tests in such a way that viral mutations only minimally evade, e.g. B. by including multiple genetic targets. The FDA also advises developers to look for mutations that can alter test performance and clearly convey test restrictions.
“We observed the different variants very closely from the start,” says Palani Kumaresan, Head of Research and Development at Roche Diagnostic Solutions. At this time, the company has no plans to change its test. The E gene signal is Pan-Sarbecovirus, the virus subgenus that contains both SARS-CoV and SARS-CoV-2. A SARS-CoV-2 specific signal comes from Orf1ab. “If we combine these two, we don’t see any effects,” says Kumaresan. Nevertheless, Roche is monitoring coronavirus variants for impaired performance and has started a separate SARS-CoV-2 variant test for research purposes.
The U.S. Centers for Disease Control and Prevention (CDC) regularly monitor primers and probes for their COVID-19 diagnostic panel and multiplex test for flu and COVID-19, according to a CDC media representative The scientist by email. CDC is also actively tracking and characterizing coronavirus variants through genomic surveillance efforts.
“As long as the virus is constantly changing – which is a natural thing – you just have to keep monitoring,” says Vogels. “When we have these studies out there looking at this, our tests will only get better.”
S. Tahan et al., “SARS-CoV-2 E Gene Variant Changes the Analytical Sensitivity Properties of Virus Detection Using a Commercial RT-PCR Assay”. J Clin Microbiol, doi: 10.1128 / JCM.00075-21, 2021.