RT-PCR based SARS-CoV-2 variant screening assays require careful quality control

Area of Science:

• Virology
• Molecular Diagnostics
• Public Health

Background:

• Emergence of SARS-CoV-2 variants of concern (VOC) with increased transmissibility and immune evasion poses a global health challenge.
• Clinical laboratories are mandated to identify and report these VOC.

Purpose of the Study:

• To evaluate the accuracy and reliability of molecular diagnostic assays used by laboratories for identifying SARS-CoV-2 VOC.
• To assess the scope of different molecular diagnostic approaches in VOC detection.

Main Methods:

• An external quality assessment scheme was implemented.
• Twenty-five laboratories participated, using various RT-qPCR-based assays.
• Laboratories analyzed five patient-derived samples and reported methods, results, and interpretations.

Main Results:

• Laboratories utilized diverse RT-qPCR assays targeting spike protein mutations characteristic of three VOC lineages.
• Detection of VOC-associated mutations at four specific sites correlated with higher correct classification rates.
• Low template copy number and additional genetic variations in samples reduced assay accuracy and confidence.

Conclusions:

• Melting-curve assays offer faster, less bioinformatically intensive VOC identification compared to sequencing.
• Accurate VOC classification depends on detecting multiple variant sites and adequate sample template (viral load).
• Some laboratories may need enhanced training for interpreting complex genetic data from evolving SARS-CoV-2 strains.