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

Abstract

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. .

Keywords: Artificial intelligent; COVID-19; High-throughput PCR; Melting analysis; Pooling; Semi-nested.

Graphical abstract

Fig. 1

Overview of the assay. A: (1) cDNA was synthesized from viral RNA and then pre-amplified using a conventional thermocycler (2.5 h); Pre-amplified cDNA samples were either (2) individually amplified or (3) pooled then amplified in a single-tube PCR reaction (1.5 h), prior to (4) Melting Analysis for the detection of SARS-CoV-2; (5) If a pool is reported as negative with SARS-CoV-2, all samples included in that pool are reported negative; (6) If a pool is positive with SARS-CoV-2, the pre-amplified cDNA samples included in the positive pool are sub-pool tested and/or individually tested following the plan created by the cloud-based application. B: Principles of the heptaplex semi-nested PCR, which includes the pre-amplification and melting semi-nested PCR reaction; Fo: outer forward primer, Ro: outer reverse primer, Fi: inner forward primer.

Fig. 2

Detection of SARS-CoV-2 RNA with the semi-nested RT-PCR assay. (A) Representative raw melting spectra resulted from 2 samples positive with SARS-CoV-2 RNA (blue lines: Positive samples), 6 samples negative with SARS-CoV-2 RNA (orange lines: Negative samples), positive control (red line) and negative control (green line); and (B) Output generated with the cloud-based application that interprets the melting spectra collected from each well of the realtime PCR instrument.