A sensitive multiplex RT-qPCR assay to detect SARS-CoV-2 in respiratory samples

We developed an in-house RT-qPCR assay for the detection of SARS-CoV-2 from different sample types. Novel primer and probe sets were designed to amplify 2 regions of the nucleocapsid (N) gene. Two dual-target multiplex assays were then evaluated for performance in detection of SARS-CoV-2 in known samples. An assay combining one novel primer pair for N (N6), a published region for the envelope protein (E) and an extraction control target (MS2), was found to have good efficiency and a low limit of detection, and to be sensitive and specific for SARS-CoV-2 in a sample set of known positive and negative clinical samples. Assay performance was maintained for the main variants of concern (Alpha, Delta, Gamma and Omicron BA.1, BA.2, BA4, JN.1). The assay was used to test for SARS-CoV-2 in throat swab samples collected in schools from teenagers in the UK as part of an observational study of two Meningococcal B Vaccines in Jan-March 2020. The low prevalence of positives identified (0.44-0.47% in two weeks of March 2020) suggests that there was minimal circulation of SARS-CoV-2 in UK schools prior to the first lockdown. The N6/E/MS2 assay has since been used to detect SARS-CoV-2 in samples from multiple studies. Continual monitoring will ensure that performance of this novel multiplex assay is maintained as novel sub-variants emerge.