Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis

Background Asymptomatic transmission was found to be the Achilles’ heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. Methods A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. Results The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83–100) and a specificity of 99.87% (95% CI: 99.55–99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. Conclusion The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity..