Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Area of Science:

• Virology
• Molecular Biology
• Clinical Diagnostics

Background:

• The COVID-19 pandemic necessitates stringent safety measures in medical laboratories.
• Inactivation of SARS-CoV-2 in clinical samples is crucial for protecting laboratory personnel.
• The impact of different inactivation methods on SARS-CoV-2 RNA detection remains unclear.

Purpose of the Study:

• To assess the effect of various inactivation methods on SARS-CoV-2 RNA copy number.
• To compare the efficacy of TRIzol LS reagent and heat-based inactivation methods.
• To determine the optimal inactivation method to minimize RNA degradation for accurate testing.

Main Methods:

• Digital PCR was used to quantify absolute SARS-CoV-2 RNA copy numbers in nasopharyngeal swab samples.
• Three inactivation methods were tested: TRIzol LS reagent, 56°C heat, and high-temperature treatments (80°C, 100°C, 121°C).
• Viral RNA copy numbers were measured before and after each inactivation procedure.

Main Results:

• TRIzol LS reagent treatment reduced N gene and ORF 1ab RNA by 47.54% and 39.85%, respectively.
• 56°C heat treatment reduced N gene and ORF 1ab RNA by 48.55% and 56.40%, respectively.
• High-temperature treatments (80°C, 100°C, 121°C) caused significant RNA degradation, with near-complete loss after autoclaving or boiling.

Conclusions:

• Sample inactivation methods significantly reduce detectable SARS-CoV-2 RNA levels.
• Heat inactivation, especially at higher temperatures, leads to substantial RNA loss and potential false-negative results.
• TRIzol LS reagent is recommended for SARS-CoV-2 sample inactivation due to its minimal impact on viral RNA quantity.