Adjusting the Cut-Off and Maximum Pool Size in RT-qPCR Pool Testing for SARS-CoV-2

Area of Science:

• Molecular Biology
• Infectious Disease Diagnostics
• Public Health

Background:

• Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is crucial for detecting SARS-CoV-2 RNA and monitoring COVID-19.
• Pool testing in RT-qPCR can increase testing efficiency but introduces challenges due to sample dilution.
• Maintaining test sensitivity and accuracy in pooled samples is essential for reliable COVID-19 surveillance.

Purpose of the Study:

• To propose a methodology for determining maximum pool sizes and adjusting cycle threshold (Ct) values in RT-qPCR pool testing.
• To ensure that samples detectable in individual testing remain detectable when tested in pools.
• To enhance the consistency and reliability of RT-qPCR pool testing for large-scale COVID-19 detection.

Main Methods:

• Developed a procedure to calculate maximum pool size and adjust Ct cut-off values to compensate for dilution in pooled RT-qPCR.
• Explicitly defined the trade-off between pool size and test sensitivity.
• Validated the methodology through pool testing of adults with flu-like symptoms at a COVID-19 reference unit.

Main Results:

• The proposed methodology successfully compensated for dilution effects in pooled RT-qPCR testing.
• Samples near the limit of detection in individual tests were accurately identified in pooled tests.
• The adjusted cut-off values, dependent on pool size, maintained detection sensitivity and avoided false negatives.

Conclusions:

• The developed methodology ensures compatible detectability scales between individual and pooled RT-qPCR sample processing.
• This approach enhances the consistency of RT-qPCR pool testing, making it more reliable for large-scale COVID-19 surveillance.
• The findings support the increased use of RT-qPCR pool testing to improve the efficiency and reach of COVID-19 diagnostics.