Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens

JCI Insight. 2020 Jun 18;5(12):e139042. doi: 10.1172/jci.insight.139042.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

Keywords: COVID-19; Molecular diagnosis; Molecular pathology; Virology.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Viral / immunology
  • Antigens, Viral / isolation & purification
  • Betacoronavirus / genetics
  • Betacoronavirus / immunology
  • Betacoronavirus / isolation & purification*
  • Clinical Laboratory Techniques / methods
  • Coronavirus Infections / diagnosis
  • Coronavirus Infections / pathology
  • Coronavirus Infections / virology*
  • Formaldehyde
  • Humans
  • Immunohistochemistry
  • In Situ Hybridization
  • Mice
  • Nucleocapsid Proteins / immunology
  • Pandemics
  • Paraffin Embedding / methods
  • Pathology, Molecular / methods
  • Pneumonia, Viral / pathology
  • Pneumonia, Viral / virology*
  • RNA, Viral / isolation & purification
  • Rabbits
  • Tissue Fixation / methods

Substances

  • Antibodies, Viral
  • Antigens, Viral
  • Nucleocapsid Proteins
  • RNA, Viral
  • Formaldehyde

Supplementary concepts

  • COVID-19
  • COVID-19 diagnostic testing
  • severe acute respiratory syndrome coronavirus 2