Tags

Type your tag names separated by a space and hit enter

Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens.
JCI Insight. 2020 06 18; 5(12)JI

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.

Authors+Show Affiliations

United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, USA.United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, USA.United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, USA.United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, USA.Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Fort Detrick, Frederick, Maryland, USA.United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, USA.

Pub Type(s)

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

Language

eng

PubMed ID

32379723

Citation

Liu, Jun, et al. "Molecular Detection of SARS-CoV-2 in Formalin-fixed, Paraffin-embedded Specimens." JCI Insight, vol. 5, no. 12, 2020.
Liu J, Babka AM, Kearney BJ, et al. Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens. JCI Insight. 2020;5(12).
Liu, J., Babka, A. M., Kearney, B. J., Radoshitzky, S. R., Kuhn, J. H., & Zeng, X. (2020). Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens. JCI Insight, 5(12). https://doi.org/10.1172/jci.insight.139042
Liu J, et al. Molecular Detection of SARS-CoV-2 in Formalin-fixed, Paraffin-embedded Specimens. JCI Insight. 2020 06 18;5(12) PubMed PMID: 32379723.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens. AU - Liu,Jun, AU - Babka,April M, AU - Kearney,Brian J, AU - Radoshitzky,Sheli R, AU - Kuhn,Jens H, AU - Zeng,Xiankun, Y1 - 2020/06/18/ PY - 2020/04/10/received PY - 2020/05/06/accepted PY - 2020/5/8/pubmed PY - 2020/7/1/medline PY - 2020/5/8/entrez KW - COVID-19 KW - Molecular diagnosis KW - Molecular pathology KW - Virology JF - JCI insight JO - JCI Insight VL - 5 IS - 12 N2 - 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. SN - 2379-3708 UR - https://www.unboundmedicine.com/medline/citation/32379723/Molecular_detection_of_SARS_CoV_2_in_formalin_fixed_paraffin_embedded_specimens_ L2 - https://doi.org/10.1172/jci.insight.139042 DB - PRIME DP - Unbound Medicine ER -