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.

BACKGROUND: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. METHODS: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. RESULTS: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9-132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7-334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3-158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7-129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). CONCLUSION: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.

An Upgrade on the Surveillance System of SARS-CoV-2: Deployment of New Methods for Genetic Inspection.

SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.

SARS-CoV-2 Variant Screening Using a Virus-Receptor-Based Electrical Biosensor.

SARS-CoV-2 variants are of particular interest because they can potentially increase the transmissibility and virulence of COVID-19 or reduce the effectiveness of available vaccines. However, screening SARS-CoV-2 variants is a challenge because biosensors target viral components that can mutate. One promising strategy is to screen variants via angiotensin-converting enzyme 2 (ACE2), a virus receptor shared by all known SARS-CoV-2 variants. Here we designed a highly sensitive and portable COVID-19 screening biosensor based on the virus receptor. We chose a dual-gate field-effect transistor to overcome the low sensitivity of virus-receptor-based biosensors. To optimize the biosensor, we introduced a synthetic virus that mimics the important features of SARS-CoV-2 (size, bilayer structure, and composition). The developed biosensor successfully detected SARS-CoV-2 in 20 min and showed sensitivity comparable to that of molecular diagnostic tests (∼165 copies/mL). Our results indicate that a virus-receptor-based biosensor can be an effective strategy for screening infectious diseases to prevent pandemics.

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.

Mutation-Specific SARS-CoV-2 PCR Screen: Rapid and Accurate Detection of Variants of Concern and the Identification of a Newly Emerging Variant with Spike L452R Mutation.

The emergence of more transmissible and/or more virulent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has triggered intensive genomic surveillance, which is costly and difficult to sustain operationally over the long term. To address this problem, we developed a set of four multiplex mutation-specific PCR-based assays with same-day reporting that can detect five VOC and three variants of interest (VOI), as defined in the March 2021 guidelines from the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/coronavirus/2019-ncov/). The screening results were compared to the whole-genome sequencing (WGS) and showed 100% concordance for strain typing for B.1.1.7 (n = 25) and P.1 (n = 5) variants using spike (S) mutation S-N501Y, S-E484K, and S-H69-V70del assays. The S-L450R assay, designed to detect the B.1.427/429 VOC, also identified multiple isolates of a newly emerging multiply mutated B.1.526.1 variant that is now rapidly increasing in the eastern United States. PCR approaches can be easily adopted in clinical laboratories, providing rapid screening methods to allow early detection of newly emergent variants and to efficiently triage cases for full genomic sequencing.