Researchers explore cost-effective Identification method for novel SARS-CoV-2 variants

Several novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have emerged since the inception of the pandemic. These variants often displaying enhanced infectiousness and virulence compared with wild-type strain. Whole genome sequencing is currently the gold standard in the identification and monitoring of these lineages. However, the set-up costs and operational requirements of facilities capable of processing such large sample volumes have been prohibitive, exceeding what is clinically actionable for most patients.

In a new study, recently released on the preprint server medRxiv*, a rapid and cost-effective method of detecting known variants of concern has been developed utilizing capillary electrophoresis, allowing local facilities to screen for and track SARS-CoV-2 variants of concern and implement reactionary procedures.

Proof of concept

The group selected three deletion mutations that were initially described as belonging to the B.1.1.7 (UK) variant: F144 on the spike protein, which is specific to this strain; an ORF1A deletion that is also present in several other variants of concern, making a distinction between B.1.1.7 and any of these other strains possible; and another gene common to all coronaviruses to act as a control. Capillary electrophoresis separates the molecular components of a sample based on mass and charge, in this case, determined mainly by nucleic acid chain length. As the variants of concern contain deletion mutations at these known sites, the nucleic acid sequence for this gene is slightly shorter. It thus elutes at a slightly different rate during electrophoresis.

One hundred eighty-six retrospective samples, dated from December 2020 to January 2021, were collected by the group, in addition to 466 prospective samples collected since this time in North Texas, USA. Among the retrospective samples, no variants of concern were detected, B.1.1.7 only becoming apparent by late January 2021, closely matching the reported spread of this strain through the state at this time.

During this pilot study, the group noted that the reverse primer utilized to be complimentary with, and thereby amplify, the spike protein F114 mutation overlapped with another distinct mutation to the spike protein observed in the emerging B.1.429/B.1.427 variants in California: W152C. As this mutation is a point change rather than a deletion of a base, the same strategy could not be employed to detect this strain. In this case, the group redesigned the primer to preferentially bind with wild-type SARS-CoV-2 over strains bearing this mutation by selected alterations in the primer sequence to enhance or dissuade binding, meaning that this strain could now be differentiated by the absence of complementary DNA.

Tracking variants of concern

Several mutations have been widely reported in multiple SARS-CoV-2 lineages, each having undergone convergent evolution towards a more virulent form by key mutations that enhance the affinity of the spike protein towards the angiotensin-converting enzyme 2 (ACE2) receptor, or provide some other enhancing function. N501Y and E484K are two such mutations that have repeatedly independently occurred, and the group next applied the assay towards these mutations. While being only somewhat indicative of precise lineage, this assay could be used to quickly highlight the need for a more detailed examination of a sample when one of these key reoccurring mutations is found to be present.

The authors were able to successfully distinguish between B.1.1.7, B.1.351, B.1.529, P.1, B1.427 and B.1.429 lineages using the assay, possible by a simple exchange of the primer sequence employed. As many as 40 primer pairs can be employed simultaneously against 96 specimens using the assay, allowing the screening procedure to encompass a large number of lineages and to avoid repeat testing. The discovery of highly specific characteristic markers for each lineage is the driving force behind the further development of this technology. However, widespread lineage tracking and monitoring are certain to play a large role in future COVID-19 management strategies. The development of such a convenient, low-cost assay will allow for improved data collection and monitoring.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.