Modifying a Diagnostic SARS-CoV-2 Spike PCR to Turn a Del69/70 Dropout into a Discriminatory On-Target Assay

To the Editor-in-Chief:

With great interest, we read the recent article by Zhen and Berry¹ presenting a novel first-line diagnostic quantitative PCR assay for SARS-CoV-2 detection, targeting the viral S gene. Following reports of increasing abundance of SARS-CoV-2 variants, such as VOC202012/01 in Britain and 501.V2 in South Africa, a number of single nucleotide polymorphisms and in-frame deletions within the S gene have come into the spotlight in recent weeks.² These mutations are increasingly attracting attention due to their potential consequences for infection control and vaccination programs.

Notably, Spike-del-HV69/70 (in-frame deletion) is present in 6.82% of all sequences listed in the GISAID database (351,996 entries as of January 17, 2021), including, for example, the B.1.1.7 lineage (VOC202012/01) and the Mink cluster V lineage.² Presence of the del-HV69/70 mutation leads to a complete loss of signal with Zhen and Berry's quantitative PCR assay³ due to the localization of its TaqMan probe's (Thermo Fisher Scientific, Waltham, MA) target sequence (21,759 to 21,782) spanning across the mutated region. As a result, variants like B.1.1.7 would be systematically missed by the assay. This, however, creates the opportunity to create a second TaqMan probe based on Zhen and Berry's design to target del-HV69/70 variants specifically, for example, using locked nucleic acid bases to increase melting temperature (Probe 2, 5′YakimaYellow-TGG TCC CAG (+A)(+G)A T(+A)G C(+A)T-BHQ1).

We have successfully used this modified version of the assay as part of typing panels to screen for SARS-CoV-2 variants. A protocol and clinical validation panel for a manual single-plex application can be found on the protocols.io website (https://www.protocols.io; last accessed April 29, 2021) Furthermore, we have integrated a modified version of the assay into a fully automated high-throughput multiplex application for a diagnostic multitarget SARS-CoV-2 PCR test with integrated typing functionality, taking advantage of its high analytic sensitivity in addition to the ability to differentiate for the deletion.³

Including the additional TaqMan probe (Probe 2) in Zhen and Berry's assay is a simple way to restore inclusivity of the test for del-HV69/70 variants, without making any further changes to the original primer/probe set. As an additional benefit, the dual-probe approach allows the rapid discrimination between del-HV69/70 variants and HV69/70-WT, for example, to screen for B.1.1.7 prior to sequencing. It should be noted that the detection of del-HV69/70 is not sufficient to confirm a B.1.1.7 lineage isolate. Further tests for N501Y and P681H, for example, can increase confidence in the call, and next-generation sequencing should be considered the definite result.