Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

Abstract

Background: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. Methods: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. Results: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. Conclusion: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants

“…The S-gene target dropout (due to the HV69-70 deletion) observed on the widely used TaqPath SARS-CoV-2 assay (Thermofisher) has reportedly been used for tracking the expansion of the B.1.1.7 lineage [ 6 ] and is now used again for the same purpose with the novel Omicron (Non-BA.2) variant [ 8 ]. However, due to the unspecific nature of assay dropouts and the prevalence of NTD deletions in non-Omicron variants, it is preferable to specifically detect SNPs or combinations of SNPs that are conserved within the lineage in question [ 16 ]. There now exists a wide range of lab-developed and commercial solutions for this purpose [ 12 , 13 , 14 ], although their suitability for application on a large scale varies as many available protocols are highly manual and require careful interpretation of every individual result.…”

“…A set of previously described TaqMan-assays of our group [ 15 , 16 ] was modified for the respective target regions of the Omicron variant S-gene [ 16 ]. Briefly, assays were designed using PrimerQuest software (IDT) with probes being 12–20 bp in length, containing a triplet of locked nucleic acid (LNA)-bases at the SNP location and melting temperature being adjusted by including additional LNA-bases.…”

“…By using this approach, we generated two Omicron “specific” targets (A67V + DEL69/70; and P681H + N679K). For sequence variants within the probe regions that are not covered by specific probes, blocker oligos were included in the assay [ 16 ].…”

Biosensors for Point Mutation Detection

“…Biosensors based on SPR, CRISPR/Cas, LAMP and aptamers have been also proposed for the detection of other SARS-CoV-2 point mutations ( Xi et al, 2021 ). Other approaches developed recently for detecting SARS-CoV-2 point mutations include a toolset for qPCR-based SNP detection ( Noerz et al, 2021 ) and full genome tiling array that can analyze the whole SARS-CoV-2 genome at single nucleotide resolution ( Jiang et al, 2021 ). Dual synthetic mismatches CRISPR/Cas12a (dsmCRISPR) method for the detection of D614G mutation with high specificity and sensitivity has been presented recently ( Huang et al, 2021 ).…”

Special Issue “Molecular Detection and Typing of Viruses”

“…This allowed to characterize spatio-temporal virus load dynamics of this new virus and shed some light on the pathophysiology of the infection, termed COVID-19 [4]. Molecular assays for detection of SARS-CoV-2 variants have next been developed to screen for variants of concern, with one example being published in this special issue [5]. Lately, next generation sequencing has become widely used to track down new variants and follow the evolution of this new virus [6].…”