Potential pitfalls in analysing a SARS-CoV-2 RT-PCR assay and how to standardise data interpretation

Area of Science:

• Molecular Biology
• Virology
• Clinical Diagnostics

Background:

• Real-time RT-PCR is the gold-standard for SARS-CoV-2 detection.
• High-throughput laboratories face challenges with complex and time-consuming data interpretation.
• Automated systems aim to simplify and expedite the analysis of real-time PCR data.

Purpose of the Study:

• To investigate the reliability of two real-time PCR data interpretation methods (relative threshold and baseline threshold) for SARS-CoV-2 detection.
• To determine the frequency of ambiguous results generated by the relative threshold method.
• To present a solution for simplifying and automating SARS-CoV-2 data interpretation.

Main Methods:

• An audit of 138 samples, including serial samples from 38 patients, was conducted.
• Comparison of results obtained using the relative threshold and baseline threshold methods.
• Evaluation of an automated interpretation system (pcr.ai) integrated with QuantStudio software.

Main Results:

• The relative threshold method produced unreliable results in a significant number of cases, leading to ambiguous curves.
• Ambiguous curves required manual intervention, increasing workload and potential for error.
• The baseline threshold method demonstrated greater reliability in initial assessments.

Conclusions:

• The relative threshold method is not consistently reliable for SARS-CoV-2 detection in high-throughput settings.
• Automated interpretation systems require careful validation of chosen threshold methods.
• A simplified and automated interpretation process is crucial for efficient and accurate SARS-CoV-2 diagnostics.