Cycle Threshold Probability Score for Immediate and Sensitive Detection of B.1.351 SARS-CoV-2 Lineage

Abstract

Background Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern associated with immune escape is important to safeguard vaccination efficacy. We describe the potential of delayed N gene amplification in the Allplex SARS-CoV-2 Assay (Seegene) for screening of the B.1.351 (20H/501.V2, variant of concern 2 [VOC.V2], South African SARS-CoV-2 variant) lineage. Methods In a study cohort of 397 consecutive polymerase chain reaction–positive samples genotyped by whole-genome sequencing, amplification curves of E/N/S-RdRP targets indicated delayed N vs E gene amplification characteristic of B.1.351. Logistic regression was used to calculate a VOC.V2 probability score that was evaluated as a separate screening test in an independent validation cohort vs sequencing. Results B.1.351 showed a proportionally delayed amplification of the N vs E gene. In logistic regression, only N and E gene cycle thresholds independently contributed to B.1.351 prediction, allowing calculation of a VOC.V2 probability score with an area under the curve of 0.94. At an optimal dichotomous cutoff point of 0.12, the VOC.V2 probability score achieved 98.7% sensitivity at 79.9% specificity, resulting in a negative predictive value (NPV) of 99.6% and a positive predictive value of 54.6%. The probability of B.1.351 increased with an increasing VOC.V2 probability score, achieving a likelihood ratio of 12.01 above 0.5. A near-maximal NPV was confirmed in 153 consecutive validation samples. Conclusions Delayed N vs E gene amplification in the Allplex SARS-CoV-2 Assay can be used for fast and highly sensitive screening of B.1.351.

From Delta to Omicron SARS-CoV-2 variant: Switch to saliva sampling for higher detection rate

“…Due to the high specificity of RT-PCR, participants were defined as having a SARS-CoV-2 infection when either saliva, OPS or NPS was positive. To determine the SARS-CoV-2 variant for each participant, SARS-CoV-2 whole genome sequencing (WGS) and clade calling [7] (Ct value < 25) or Allplex SARS-CoV-2 variants I (E484K, HV69/70del, N501Y) and II (K417N, K417T, L452R, W152C) Assay (Seegene, Seoul, Republic of Korea) (Ct value ≥ 25) was performed on the NPS or saliva (sample with lowest Ct value). In brief: WGS was performed using the Research Use Only AmpliSeq for Illumina SARS-CoV-2 Research Panel on Illumina MiSeq (80 samples on V2 flow cell) according to the manufacturer's standard protocol: 8 µl RNA extract was reverse transcribed using Lunascript RT SuperMix Kit (New England Biolabs, MA, USA), followed by amplification of 237 virus specific amplicons covering > 99% of the 30kb reference genome aiming at a median coverage above 500x, minimal coverage for mutation calling of 10x and variant allele frequency > 90% and a minimum of 30,000 reads per sample and a maximum of 1kb bases below minimal coverage.…”

Cov2MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein in infectious patients

“…Residual Covid-19 nasopharyngeal patient samples were obtained from the AZ Delta hospital, Roeselare, Belgium with approval of the University Hospital Ghent ethics committee (BC-09263). These samples were analysed at the clinical laboratory of the AZ Delta hospital using the Allplex 2019-nCoV RT-PCR assay from Seegene Inc. as described by De Smet et al 15 . In short, RNA was extracted from nasopharyngeal swabs using a STARMag 96 × 4 Viral DNA/RNA 200 C Kit (Seegene Technologies, Walnut Creek, CA, USA) on the Hamilton STARlet workstation, followed by real-time PCR using the Allplex SARS-CoV-2 assay.…”

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein

“…Residual Covid-19 nasopharyngeal patient samples were obtained from the AZ Delta Hospital, Roeselare, Belgium, with approval of the University Hospital Ghent ethics committee (BC-09263). These samples were analyzed at the clinical laboratory of the AZ Delta Hospital using the Allplex 2019-nCoV RT-PCR assay from Seegene Inc. 16 …”

“…Residual Covid-19 nasopharyngeal patient samples were obtained from the AZ Delta Hospital, Roeselare, Belgium, with approval of the University Hospital Ghent ethics committee (BC-09263). These samples were analyzed at the clinical laboratory of the AZ Delta Hospital using the Allplex 2019-nCoV RT-PCR assay from Seegene Inc. 16 Lyophilized recombinant N protein was reconstituted to a concentration of 0.1 μg/μL in 100 mM NH 4 HCO 3 . A 50 fmol/μL calibration standard of N was prepared in SARS-CoV-2-negative nasopharyngeal swab pools of different media, i.e., 100 mM NH 4 HCO 3 , Copan Universal Transport Medium (UTM), Bioer UTM, Sigma Virocult, eSwab, PBS, plasma, synthetic saliva (saliva substitute, donated by the University of Leicester), and patient saliva.…”