Skip to main content
Premium Trial:

Request an Annual Quote

Rutgers University Researchers Develop PCR-Based SARS-CoV-2 Assay to Detect All Known Variants


NEW YORK – Researchers at Rutgers University have developed a molecular test they say can detect all known variants of SARS-CoV-2, potentially offering a strategy to help guide treatments and track the spread of COVID-19.

David Alland and his colleagues recently published a preprint study describing the test in MedRxiv detailing how the test works with the Omicron variant, the culprit in the most recent surge of COVID-19 cases.

The test works by amplifying the sections of the spike protein of SARS-CoV-2 that have signature mutations for each variant, using RT-PCR that includes six "sloppy molecular beacons," or DNA probes that melt off of their DNA targets at different temperatures. It then uses the pattern of how they melt to determine which mutations, and thus which variant, are present. Sloppy molecular beacons, or SMBs, aren't completely dependent on the sequences they hybridize to, making them more effective against new mutations that could arise, said Alland, chief of infectious diseases and director of the Public Health Research Institute and the Center for COVID-19 Response and Pandemic Preparedness at Rutgers University New Jersey Medical School.

To run the test, the virus's RNA is extracted, reverse transcribed, and amplified with PCR in the presence of the six SMBs and a control; the beacons are then melted off, Alland said. The SMBs have been labeled with different colored fluorescence, and as the beacon melts off the fluorescence fades. Once the fluorescence disappears, it indicates the temperature at which the beacon has melted off, which then determines the variant through data analysis. 

Excluding samples with indeterminant results, the test showed 100 percent sensitivity and specificity compared to sequencing, according to the MedRxiv study.

After extracting RNA, the total process takes about an hour and 15 minutes, Alland said. While RNA extraction methods and times can vary, he said the assay could be used as a "reflex test" on positive samples that have likely already had the RNA extracted.

For the MedRxiv study, Alland's team preferred the Roche LightCycler 480 PCR instrument, but he said the test has also been validated "on a number of instruments," including Bio-Rad Laboratories' CFX96, Qiagen's Rotor-Gene Q, and Thermo Fisher Scientific's ABI 7500. The test shouldn't have a "substantially higher" cost than other PCR tests, he noted.

Another benefit of the test is that the sample type doesn't matter, he said — saliva, nasal, and nasopharyngeal swabs all worked with the assay. 

Alland and his team originally published a study in the Journal of Clinical Microbiology describing an earlier version of the test in September 2021. The work for that paper, however, was conducted before the emergence of either the Delta or Omicron variant. To adapt the test to detect the Delta mutations, the team added two additional probes to the test, and for Omicron it added updated temperature signatures, Alland said.

If another new variant arises, he said that he's hoping that the key mutations will be in the same regions and the same probes will pick them up, so the only change necessary will be adding another temperature signature. He noted that if that doesn't happen, the team could make new probes for the test or swap probes out "as needed," although he's "hopeful we've seen the last of the major surges."

His team is also working on adding another probe for the test that would work on the N gene in SARS-CoV-2, which would allow it to be used as a diagnostic test for the virus, he said. Currently, the test is only being used once a sample has been identified as positive for SARS-CoV-2. 

Alland's lab originally developed a similar method of detecting bacterial pathogens from blood cultures, and then used the method to create a 10-color drug-resistant tuberculosis test for Cepheid. Both tests have been approved by the World Health Organization, and the Cepheid test is CE-IVD marked.

Alland said his team took the experience of developing those tests and applied the method to SARS-CoV-2. Although his lab has worked with Cepheid and he receives grants and royalties from the Danaher subsidiary, he said this SARS-CoV-2 test wasn't designed for Cepheid, but rather "for whoever needed it," which is why it was tested on different instruments.

He added that he doesn't have current plans for partnering with any company to commercialize the test but that he'd "be happy to do that" if there's interest. Alland added that the team hopes to be able to offer its testing services with the assay to "local institutions." The test could also be adapted for use in another lab, he said. 

There's a benefit in publishing a preprint about how the test works, he said — it can be updated for other variants as needed, which Alland said the team will do when and if necessary. The authors do intend to submit the preprint for peer review, he added.

The assay isn't being used clinically yet, he said, but the team has worked with partners at Rutgers who have applied to the New Jersey Department of Health to use it as a laboratory-developed test in the state.

"The idea really was to get this test out quickly because … these variants come through so quickly that as soon as you develop a variant test, it becomes obsolete," he said. 

But one potential question is whether the test will still have clinical utility if another major surge doesn't occur. Alland said he's hopeful that a combination of vaccine-induced immunity and natural immunity will mean that another variant doesn't arise, or that a new variant will spread less quickly, which would mean the test's utility may "be used up." But it will be "there and ready" if it's needed, making it easier to track and respond to potential new variants, he said.

David Perlin, CSO and senior VP of Hackensack Meridian Health's Center for Discovery and Innovation who has also developed a test for SARS-CoV-2 variant screening, said that the identification of variants has two key clinical applications: to better inform clinicians about the most effective treatment, as "variants of concern like Delta and Omicron are less sensitive or resistant to certain first-line antibody cocktail regimens"; and to provide data on the spread of variants in the community and in the acute care setting.

Whether the test will become obsolete, Perlin said that molecular tests of this kind "are robust and new markers can be added" as new variants emerge. 

He noted that the researchers should "probably increase their sample size to better assess negative and positive predictability," but added that they have "professionally designed" their platform "for sensitivity and specificity."