Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, necessitates scalable diagnostic methods.
• Real-time RT-PCR is the primary diagnostic tool, but test kit availability can be a bottleneck.
• Optimizing existing protocols is crucial for efficient global diagnostics.

Purpose of the Study:

• To evaluate the compatibility of WHO-recommended real-time RT-PCR assays with different PCR chemistries.
• To assess the impact of reduced reaction volumes on SARS-CoV-2 detection sensitivity.
• To enhance the cost-effectiveness and resource efficiency of COVID-19 diagnostics.

Main Methods:

• Selected three WHO-listed real-time RT-PCR assays for SARS-CoV-2 detection.
• Investigated compatibility with three different PCR chemistries.
• Reduced reaction volumes by up to 50% and tested clinical samples and viral dilutions.

Main Results:

• Nearly identical quantification cycle (Cq) values were observed across all assay/chemistry combinations using cultured virus.
• Consistent results were obtained for negative and high-to-medium viral load samples across all tested combinations.
• Inconsistent results were noted for very low viral loads (Cq > 36), without a clear preference for specific viral targets (E, RdRp, N) or chemistries.

Conclusions:

• Reducing reaction volume and altering PCR chemistry do not compromise SARS-CoV-2 detection sensitivity.
• The findings support increased flexibility and cost-effectiveness in COVID-19 diagnostics.
• Optimized protocols can help overcome bottlenecks in large-scale SARS-CoV-2 testing.