Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

Accurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.