Importance of sample input volume for accurate SARS-CoV-2 qPCR testing

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Medicine

Background:

• Nucleic acid testing, particularly real-time quantitative reverse transcription PCR (qPCR), is the primary method for detecting SARS-CoV-2.
• Current qPCR assays exhibit limitations in reliably detecting low viral loads (Ct-value ≥ 35), leading to potential false-negative results.
• The inconsistency in low viral load detection is attributed to the random sampling of target molecules.

Purpose of the Study:

• To investigate the impact of sample input volume on the performance of SARS-CoV-2 qPCR assays.
• To elucidate the statistical basis (Poisson distribution) for false-negative outcomes in low viral load detection.
• To demonstrate a method for improving the accuracy and consistency of COVID-19 nucleic acid testing.

Main Methods:

• Utilized a Poisson distribution model to analyze the probability of sampling zero target molecules in qPCR.
• Increased the purified RNA input volume from 2 μL to 10 μL in SARS-CoV-2 qPCR assays.
• Validated the effect of increased input volume on a SARS-CoV-2 qPCR assay with a limit of detection (LOD) of 300 copies/mL using clinical samples.

Main Results:

• The probability of sampling zero RNA molecules decreased significantly with increased input volume, from 36.79% to 0.67% at 0.5 copy/μL RNA concentration.
• Increasing purified RNA input from 2 to 10 μL reduced false-negative qPCR results in clinical samples (Ct ≥ 35) by 50%.
• Enhanced sample input volume demonstrably improved the consistency and accuracy of detecting low viral loads.

Conclusions:

• False-negative results in SARS-CoV-2 qPCR are linked to the random chance of not sampling target molecules, especially at low viral concentrations.
• Increasing the sample input volume is a practical strategy to mitigate sampling errors and enhance the reliability of low viral load detection.
• Optimizing sample input volume is crucial for improving the overall consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2.