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

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.

Figure 1. Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets.

(A and B) In comparison to uninfected control FFPE cell pellets (A and C), SARS-CoV-2 S (brown, B) and SARS-CoV-2 NP (brown, D) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). (E) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A–D; 20 μm in inset of E; and 10 μm in E.

Figure 2. Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets.

(A and B) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets (B), but not in uninfected control FFPE cell pellets (A). (C and D) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets (D), but not in uninfected control FFPE cell pellets (C). (E and F) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets (E), but not in uninfected control FFPE cell pellets (F). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

Figure 3. Detection of SARS-CoV-2 replication in FFPE cells using mFISH.

(A and B) Compared with uninfected control (A), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA (B). Nuclei are stained blue (DAPI). Scale bars: 20 μm.

Figure 4. Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section.

(A and B) Compared with uninfected control FFPE cell pellets (A), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section (B). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.