Rapid Detection of SARS-CoV-2 Variants: A Multiplexed RT-PCR Approach

Microbiologist studying coronavirus with microscope

The ongoing COVID-19 pandemic caused by SARS-CoV-2 continues to present new challenges, especially with the emergence of viral variants like Omicron and Delta. These new variants can spread more efficiently, evade immunity from past infection or vaccines, and resist treatment with antibody therapies. Therefore, the ability to quickly screen confirmed SARS-CoV-2 cases to identify specific variants is crucial for both clinical management of individual patients as well as guiding public health policy decisions.

In response to this need, researchers have turned to polymerase chain reaction (PCR) techniques that can rapidly detect key viral genomic mutations that differentiate emerging variants from one another. In particular, multiplex RT-PCR assays allow simultaneous and efficient screening for mutations across different parts of the SARS-CoV-2 genome. The Mantacc-sponsored University Medical Center Hamburg-Eppendorf (UKE) research team recently developed and evaluated such a multiplexed PCR-based assay to identify the variants Delta and Omicron for high-throughput use.

Multiplex Assay Design

The assay detects four gene targets in the SARS-CoV-2 spike protein using differential fluorescent probes, including two specific to Omicron, one for Delta, and a fourth conserved target. By using special locked nucleic acids (LNAs) in the probes, they can distinguish single nucleotide polymorphisms (SNPs) between variants at specific locations involved in ACE2 receptor binding, furin cleavage activation, and epitopes for neutralizing antibodies.

Analytic and Clinical Validation

In analytical testing with quantitated cultured Omicron virus, the PCR assay detected samples down to 19-105 IU/mL depending on the target. It correctly identified Omicron, Delta and other variant specimens among 225 clinical respiratory swabs sequenced by next-generation sequencing (NGS), confirming its reliable detection of emerging lineages. All negative controls and non-SARS-CoV-2 virus samples tested negative, supporting the multiplex PCR’s specificity.

Future Considerations

While a promising option for high-volume SARS-CoV-2 variant screening, multiple key challenges exist. Novel Omicron sublineages like BA.2 lack certain target mutations detected in the current assay, requiring frequent re-validation as the virus evolves overtime. Complementary NGS is still needed for whole genome confirmation and identification of new mutations. Sample handling and workflow requirements also constrain the scalability of PCR typing versus sequencing approaches. Nonetheless, PCR flexibility and automation may make it an attractive strategy for initial binary variant identification when treatments like monoclonal antibodies show efficacy differences across lineages.

Therefore…

The urgent need to track SARS-CoV-2 variants calls for rapid, scalable and automated screening tools. Multiplexed RT-PCR methods can reliably detect emerging viral lineages in clinical samples by targeting characteristic genomic mutations with sensitive fluorescent probes. While sequencing remains vital for surveillance, high-throughput PCR typing assays provide actionable lineage data to guide time-sensitive clinical and public health decisions in fighting the ongoing pandemic. Continued variant evolution however demands consistent reappraisal of mutation detection targets and interpretational caveats for this emerging molecular strategy.

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