Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads

Clin Chem. 2020 Jun 1;66(6):794-801. doi: 10.1093/clinchem/hvaa091.


Background: Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.

Methods: We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT.

Results: Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.

Conclusion: Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; false negative; nucleic acid test; thermal inactivation.

MeSH terms

  • Betacoronavirus / chemistry*
  • COVID-19
  • COVID-19 Testing
  • COVID-19 Vaccines
  • Clinical Laboratory Techniques / methods
  • Coronavirus Infections / diagnosis*
  • False Negative Reactions
  • Feces / virology
  • Guanidine / chemistry
  • Hot Temperature*
  • Humans
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • RNA, Viral / analysis*
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / statistics & numerical data
  • SARS-CoV-2
  • Specimen Handling / methods
  • Sputum / virology
  • Time Factors
  • Viral Load*
  • Virus Inactivation / drug effects


  • COVID-19 Vaccines
  • Covid-19 aAPC vaccine
  • RNA, Viral
  • Guanidine