Researchers have developed a PCR test that uses molecular beacons to diagnose a COVID infection and the specific variant causing the infection.

The research—conducted by investigators from ResearchPath LLC and their collaborators at Rutgers University—appears in The Journal of Molecular Diagnostics, published by Elsevier. Their methodology is openly available so that it can be replicated by any facility that can run a PCR test.

“It is extraordinary to see that SARS-CoV-2 was not a monolithic infection with a predictable set of clinical features, but rather an ever-evolving disease for which the different strains produce unique clinical features that affect testing, symptoms, and even which organ systems can be attacked,” says lead investigator Sanjay Tyagi, PhD, Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ, USA.

Identifying specific COVID variant strains via a PCR test reveals important information such as the length of incubation period, length of contagious period, transmissibility, pathogenicity, and even changes in the predominant symptoms.

Using real-time PCR probes designed by Rutgers University and already used around the world for many purposes, Rutgers designed the Rutgers-RP RT-PCR assay to detect mutations in COVID that have been shown to increase immune escape, avoid neutralization, and increase transmissibility. They pioneered the use of molecular beacons to identify specific genetic mutations. Molecular beacons are hairpin-shaped molecules that can be designed to selectively bind to a specific mutant sequence, avoiding wild-type sequences that often differ by a single nucleotide.

“Knowing that a highly contagious and dangerous strain is emerging in a local community could inform policymakers to initiate safety measures to limit spread,” says co-investigator Ashley Hill, MD, ResourcePath LLC, Sterling, VA, USA. “It can also serve as an early warning system for healthcare systems that need to plan for surges in ER visits and ICU care. Knowing which strain has infected a person can also help determine which treatments would be most beneficial.”

Nine COVID mutations were selected for the PCR testing, and the beacon for each has differently colored dyes. Every original variant of concern—alpha, beta, gamma, delta, and omicron—has a unique combination of these mutations. and when the beacon binds to its target molecule, its distinct color can be detected by the assay.

Each beacon was tested individually to confirm its specificity to the assigned mutation. Then, the beacons were combined into a multiplex assay and tested by RT-PCR on 26 SARS-CoV2–positive patient samples that had previously been tested and identified with deep sequencing.

Researchers report that the test is also very adaptable. When omicron emerged, the investigators were able to design a beacon in less than a month to identify a mutation that is unique to omicron and is important for immune evasion. The investigators identified the omicron variant in 17 of 33 additional patient samples that had been previously tested, and the results were 100% in agreement.

“The tools we developed to track and identify new variants will be useful for this pandemic and for any unforeseen viruses or pathogens that may arise going forward,” says lead author Ryan J. Dikdan, BS, Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ, USA.

Featured image: Each hairpin shaped molecular beacon has a specific color and fluoresces when it binds to its target genetic mutation. Photo: Salvatore Marras