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 is high.
• Many labs lack capacity for kit-free or manual SARS-CoV-2 testing protocols.
• Need for standardized, implementable SARS-CoV-2 detection workflows.

Purpose of the Study:

• To evaluate commercially available RNA extraction kits and one-step RT-qPCR reagents for SARS-CoV-2 detection.
• To assess the reliability of detecting viral N and RdRP genes.
• To determine the efficacy of heat treatment for SARS-CoV-2 inactivation in clinical samples.

Main Methods:

• Comparison of three RNA extraction kits (QIAamp, RNAdvance, Mag-Bind) and three one-step RT-qPCR reagents (FastVirus, qPCRBIO, Luna).
• Utilized primer-probes targeting SARS-CoV-2 N and RdRP genes.
• Performed plaque assays and qPCR on heat-treated nasopharyngeal swabs (original and B.1.1.7 variants).

Main Results:

• RNA extraction methods yielded comparable results; Beckman showed better performance with tested primer-probes.
• Luna reagent demonstrated highest sensitivity; overall reagent differences were not significant.
• N gene detection was more reliable than RdRP, especially in low viral load samples.
• Heat treatment (70°C or 90°C) effectively inactivated SARS-CoV-2 with minimal impact on qPCR sensitivity.

Conclusions:

• Established multiple SARS-CoV-2 testing pipelines suitable for laboratories lacking CL-3 facilities.
• Heat inactivation protocol allows for safe, portable SARS-CoV-2 testing.
• Provided freely available protocols and SOPs to facilitate implementation in other labs.