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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.

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