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

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Assays

Background:

• Asymptomatic transmission of COVID-19 challenged symptom-based screening.
• Global shortages in molecular reagents and low throughput of PCR facilities limited pandemic containment.
• Efficient mass testing strategies were urgently needed.

Purpose of the Study:

• To develop a novel assay for high-throughput SARS-CoV-2 RNA detection.
• To address limitations in molecular reagent supply and PCR facility capacity.
• To improve COVID-19 diagnostic testing efficiency.

Main Methods:

• Established a novel semi-nested, heptaplex (7-plex) reverse transcription PCR (RT-PCR) assay.
• Utilized melting analysis and an artificial intelligence algorithm for SARS-CoV-2 RNA detection.
• Evaluated analytical and clinical performance using synthesized and clinical samples.

Main Results:

• Achieved a limit of detection (LOD) of 7.2 copies/reaction for individual testing.
• Demonstrated high clinical performance: 100% sensitivity and 99.87% specificity.
• Enabled 96-sample pooled testing without compromising sensitivity, allowing up to 8,820 samples per 96-well plate.

Conclusions:

• The developed RT-PCR assay offers highly multiplexed (7-plex) detection of SARS-CoV-2.
• The assay facilitates 96-sample pooled testing, significantly enhancing diagnostic capacity.
• This approach provides a viable solution for large-scale COVID-19 screening.