key: cord-1039989-eb74kjs7 authors: Nörz, Dominik; Grunwald, Moritz; Olearo, Flaminia; Fischer, Nicole; Aepfelbacher, Martin; Pfefferle, Susanne; Lütgehetmann, Marc title: Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7 date: 2021-06-12 journal: J Clin Virol DOI: 10.1016/j.jcv.2021.104894 sha: 6b9b7dd512c9919c2649d72b3e01094151cf372b doc_id: 1039989 cord_uid: eb74kjs7 BACKGROUND: New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. METHODS: A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. For clinical performance, a total of 176 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and next generation sequencing as gold standard. RESULTS: The multiplex assay was highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 6.16 cp/ml (CI: 4.00 – 8.31). LoDs were slightly higher for detection of the HV69/70 deletion (85.92, CI: 61 – 194.41) and the N501Y SNP (105.99 cp/ml, CI: 81.59 – 183.66). A total of 176 clinical samples were tested with the assay, including 50 samples containing SARS-CoV-2 of the B.1.1.7 lineage, one containing B.1.351 and 85 non-B.1.1.7/B.1.351 lineage, of which three also harbored a HV69/70 deletion. All were correctly identified by the multiplex assay. CONCLUSION: We describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of the del-HV69/70 and N501Y mutations that can distinguish between B.1.1.7 and other lineages. The assay allows for high-throughput screening for currently relevant variants in clinical samples prior to sequencing. Background: New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. Methods: A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and A number of novel SARS-CoV-2 variants that emerged in fall 2020, some of which show increased transmissibility and possible immune escape, are increasingly attracting worldwide attention (1, 2). The B.1.1.7 lineage (VOC202012/01, 501Y.V1) first emerged in southern England and is notable for an unusually high number of mutations with no direct common ancestor compared to previous sequences (3, 4) . In particular, non-synonymous spike-gene mutations such as the N501Y SNP (single nucleotide polymorphism) may lead to changes in receptor binding properties (5) . Another variant of concern, B.1.351 (501Y.V2) first described in south Africa, are characterized by the same spike-gene mutation, along with E484K and others (6) . Both have largely replaced previously circulating lineages in their respective areas of origin, indicating improved host adaptation and immune evasion. As a result of increased awareness in public health and science institutions, there has been a rapidly growing demand for whole genome sequencing worldwide in order to recognize and map the spread of these emerging variants. In this context, the speed and scalability of fully automated RT-PCR can be highly beneficial to pre-screen samples for relevant mutations. The aim of this study was to create and validate a high-throughput first-line screening assay with the built-in ability to discriminate between relevant SARS-CoV-2 variants, most importantly B.1.1.7, on a fully automated sample-to-result PCR-platform (7) . Inhouse assays have previously been used successfully for SARS-CoV-2 detection and diagnostics with this system via its open mode (Cobas Omni Utility Channel) (8). The basic rationale of the multiplex assay was to combine two highly sensitive diagnostic SARS-CoV-2 PCR assays with additional assays to simultaneously detect relevant mutations. The SC2-assay by the 5 US CDC (N-gene, SC2-N) was modified to serve as Pan-SCoV2 target (9) . A publicly available diagnostic S-gene assay by Zhen et al. serves as second target (SC2-S) (10), while also featuring a drop-out phenomenon in the presence of a HV69/70 deletion due to probe location. An additional Taqman-probe (Probe-2, S-Del) was created to match the mutated sequence and allow differentiation for wild-type and del-HV69/70. An additional assay was designed (using Beacon designer and PrimerQuest software) and integrated into the multiplex to detect the N501Y SNP. See The cobas 6800/8800 internal control (IC) is a spike-in (packaged) RNA target, which is automatically added by the system during extraction. MMRX-R2-reagent already contains the internal control assay by default; the respective sequences are not disclosed by the manufacturer. The IC acts as a full process control in the same way as in commercial cobas 6800/8800 IVD tests manufactured by Roche. Primer/Probe Sequence (5' -3') Conc. [nM] Inclusivity Ref. for the Accuplex verification panel as stated above. Of the 136 SARS-CoV-2 positive sample-set, all were correctly identified by the SCOV2_VAR_UCT. LoD, Limit of Detection; SNP, single nucleotide polymorphism; RBD, receptor binding domain; IC, internal control; IVD, in-vitro diagnostic; RFI, relative fluorescence increase; CI, confidence interval; NGS, next generation sequencing. New variant of SARS-CoV-2 in UK causes surge of COVID-19 Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking epidemiological and genetic data Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion H69/V70 Molecular Mechanism of the N501Y Mutation for Enhanced Binding between SARS-CoV-2's Spike Protein and Human ACE2 Receptor Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa Clinical evaluation of the cobas SARS-CoV-2 test and a diagnostic platform switch during 48 hours in the midst of the COVID-19 pandemic Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection Pushing beyond specifications: Evaluation of linearity and clinical performance of a fully automated SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70 ML received speaker honoraria and related travel expenses from Roche Diagnostics.All other authors declare no conflict of interest.