Novel RT-PCR may help detect SARS-CoV-2 mutations tied to VoC

Audrey Abella
12 Oct 2021

The Spike single-nucleotide polymorphism (SNP) assay, a single-reaction multiplex real-time RT-PCR, provides fast, inexpensive, and sensitive detection of specific mutations associated with SARS-CoV-2 variants of concern (VoC), according to data presented at ID Week 2021.

With the emergence of different SARS-CoV-2 variants, it is important to maintain detection and surveillance of these variants to mitigate their negative consequences (eg, increased transmissibility, monoclonal antibody escape), said presenting author Maxwell Su from the Emory University School of Medicine, Atlanta, Georgia, US. “[Although] WGS is the current reference standard … it can take a lot of time and requires a lot of resources, which is problematic because a lot of sequencing is needed to track these variants.”

“Broadly accessible and inexpensive assays are thus needed to enhance variant surveillance and detection globally. [As such,] we developed and validated the Spike SNP assay to detect specific mutations associated with VoC,” said Su.

“[W]e also wanted an assay that can be easily modified to account for new, emerging variants [to] allow us to exploit the benefits of real-time RT-PCR, including an increased capacity for testing, a lower time for test, and less resources [compared with] sequencing,” he continued.


Analytical evaluation

Using the Spike SNP assay, signals of characterized variant strains – B.1.1.7, B.1.525, and P.1 – were identified across three target SNPs (ie, E484K, N501Y, and 417K). Signals for B.1.1.7 were found in both 417K and N501Y (linear range, 3.3–6.3 GE/mL), while signals for B.1.525 were found in 417K and E484K (linear range, 3.2–6.2 GE/mL). P.1 signals were seen in 484K and N501Y. [ID Week 2021, abstract 148]

The three target SNPs were detected throughout the dilution series, with the 95-percent lower limit of detection ranging from 2.46 to 2.48 log10 GE/mL which, according to Su, “constitutes a very sensitive assay”.


Clinical evaluation

A total of 253 residual nasopharyngeal samples that had previously tested positive for SARS-CoV-2 were evaluated. Of these, 238 were positive in the Spike SNP assay (n=220 and 18 had SARS-CoV-2 N2 Ct of <30 and ≥30, respectively). All samples were positive in another assay (ie, N2RP).

Samples that tested negative had a much higher Ct in the N2RP than in the Spike SNP assay. “[This] makes sense, as the higher Cts in the N2 samples means that there was much less genomic material for the Spike SNP assay to pick up,” Su explained.


Enhanced detection, surveillance

Though not intended for use as standalone replacement for sequencing, the Spike SNP assay may be used to select specific samples for sequencing. “[This] may help lighten the load in regions without ready access to sequencing infrastructure, time, or money to conduct sequencing at a high rate,” said Su. It may also be of benefit to regions with sequencing infrastructure but with reagent shortages.

The Spike SNP assay may also impact clinical decision making. For instance, E484K has been shown to inhibit activity of bamlanivimab. “[If we can] use our assay to detect E484K in a patient, we may be able to inform treatment decisions based on which monoclonal antibody to use in the future,” said Su.

“Our assay also shows greater potential for future customization in terms of new variants,” Su continued. “[I]f a new variant pops up with different mutations … we can simply create new unmodified probes to incorporate into our multiplex to detect this mutation in the future.”

“[Taken together,] if implemented across laboratories offering SARS-CoV-2 testing, [the Spike SNP assay] could greatly increase capacity for variant detection and surveillance globally,” concluded Su.


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