Validation of a molecular assay to detect SARS-CoV-2 in saliva

ABSTRACT

The New Zealand Microbiology Network (NZMN) has recommended that any saliva test would need to be validated locally using well characterised samples that were positive for SARS-CoV-2 and that testing be performed in a diagnostic laboratory accredited by International Accreditation New Zealand (IANZ) and aligned with the ISO 15189 quality framework.10 NZMN noted that the accuracy of saliva tests is reliant upon the methods used for saliva collection, the extraction steps employed for the viral RNA and the commercial kit utilised.10 In September 2020, we established an international collaboration with investigators at the University of Illinois Urbana-Champaign (UIUC), USA, who had developed a simple and rapid direct sali-va-to RT-qPCR assay for the detection of SARS-CoV-2.11 The assay omits the common RNA extraction step and utilises a modifed method of a commercially available RT-qPCR kit, thus avoiding reagent competition and supply-chain issues. [...]the subsequent addition of a detergent-buffer mix overcomes the drawback of saliva viscosity. The kit includes primers and probes specifc to the SARS-CoV-2 nucleocapsid (N), spike (S) and replication (ORF1ab) genes and a spike-in bacteriophage (MS2) gene, with modifca-tions to the manufacturer's instructions, as previously published.11,13 Controls included a positive control (TaqPath™ COVID19 Control;ThermoFisher Scientifc TaqPathTM COVID-19 Combo kit) at 25 copies per µL, negative control (UltraPure dH2O) and no-template control (heat-inactivated saliva;collected in-house). All nasal swabs (defned as both nasopha-ryngeal and mid-turbinate) were processed in the clinical pathology laboratory at the University of Illinois Chicago Hospital using an FDA EUA reference method for detection of SARS-CoV-2, the Abbott RealTime SARS-CoV-2 assay performed on the Abbott m2000 System with a limit of detection of 2,700 NDU per mL.13 Saliva samples that had been heat-inactivated at 95°C for 30 min in UIUC were divided into aliquots.