Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene

Area of Science:

• Clinical diagnostics
• Molecular biology
• Virology

Background:

• Accurate detection of SARS-CoV-2 viral RNA is critical for diagnosing and managing COVID-19.
• Validation of molecular assays is essential for reliable diagnostic testing.

Purpose of the Study:

• To clinically validate a reverse transcription PCR (RT-PCR) assay targeting the SARS-CoV-2 nucleocapsid (N1) gene.
• To optimize nucleic acid extraction procedures using endemic coronaviruses and recombinant viral controls.

Main Methods:

• Optimization of off-board lysis and automated nucleic acid extraction.
• Comparison of recovery rates for endemic coronaviruses (OC43, NL63) and SARS-CoV-2 RNA using different control materials.
• Clinical validation of the RT-PCR assay for sensitivity, specificity, accuracy, and precision.

Main Results:

• Nucleic acid extraction showed variable recovery of endemic coronaviruses but reliable recovery of SARS-CoV-2 RNA.
• The RT-PCR assay achieved 100% in silico sensitivity and specificity, with 100% accuracy across 75 samples.
• Precision studies demonstrated 100% concordance, and the limit of detection was determined to be 264 copies/mL.

Conclusions:

• The validated SARS-CoV-2 RT-PCR assay exhibits suitable characteristics for Emergency Use Authorization.
• Endemic coronavirus controls aided in optimizing the extraction process.
• Recombinant virus in a protein coat serves as an appropriate control for assay validation when live or attenuated viruses are unavailable.