Boosting SARS-CoV-2 detection combining pooling and multiplex strategies.

Area of Science:

• Molecular Biology
• Virology
• Public Health

Background:

• Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the standard for SARS-CoV-2 detection but faces challenges with long turnaround times and high costs.
• Increased demand for SARS-CoV-2 testing, particularly for monitoring vaccinated populations against emerging variants, necessitates strategies for enhanced capacity and cost-efficiency.

Purpose of the Study:

• To evaluate the feasibility and effectiveness of RT-qPCR sample pooling strategies for SARS-CoV-2 detection.
• To assess the impact of pooling on test sensitivity, cost savings, and overall testing capacity.

Main Methods:

• In-silico statistical modeling and validation using specimen samples from a mass testing program.
• Evaluation of RT-qPCR pooling in simplex and multiplex assays with varying pool sizes (up to 32 samples).
• Analysis of cost savings and reagent consumption associated with pooling strategies.

Main Results:

• High test sensitivity was maintained when pooling up to 16 or 8 samples in simplex assays.
• Pooling 4 or 8 samples in a triplex RT-qPCR assay (targeting N and E genes) was feasible without sensitivity loss.
• A cost saving of 51.5% was achieved in a mass testing program, even accounting for individual pool re-testing.

Conclusions:

• RT-qPCR pooling, especially in multiplex assays, offers a viable strategy for cost-effective mass SARS-CoV-2 testing.
• This approach significantly increases testing capacity and reduces reagent consumption, aiding in pandemic control.
• Pooling strategies are essential for managing high testing demands and monitoring viral spread effectively.