Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

Area of Science:

• Molecular Biology
• Virology
• Clinical Diagnostics

Background:

• Global demand for SARS-CoV-2 detection reagents necessitates accessible testing solutions.
• Many laboratories lack the capacity for kit-free or manual SARS-CoV-2 RNA extraction and detection.
• Developing standardized, implementable workflows is crucial for expanding testing capabilities.

Approach:

• Compared commercial RNA extraction kits (QIAamp, RNAdvance, Mag-Bind) and one-step RT-qPCR reagents (FastVirus, Luna, qPCRBIO).
• Evaluated primer-probe sets for SARS-CoV-2 N and RdRP genes, assessing sensitivity with varying viral loads.
• Validated heat inactivation of nasopharyngeal swabs (70-90°C) for SARS-CoV-2 inactivation using plaque assays and qPCR sensitivity.

Key Points:

• All tested RNA extraction methods yielded comparable results.
• FastVirus and Luna reagents demonstrated the highest sensitivity in RT-qPCR.
• Detection of the viral N gene was more reliable than RdRP, especially for low viral titers.
• Heat treatment effectively inactivated SARS-CoV-2 (original and B.1.1.7 variants) with minimal impact on qPCR sensitivity.
• SARS-CoV-2 testing can be performed in settings lacking high-containment (CL-3) facilities.

Conclusions:

• Multiple SARS-CoV-2 testing pipelines using commercially available reagents were established and validated.
• Heat inactivation of clinical samples offers a safe and effective method for sample preparation.
• The study provides freely available protocols and SOPs to facilitate widespread implementation of SARS-CoV-2 detection.