Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens.

Autor:	Liu J; United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA., Babka AM; United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA., Kearney BJ; United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA., Radoshitzky SR; United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA., Kuhn JH; Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National, Institutes of Health, Fort Detrick, Frederick, MD 21702, USA., Zeng X; United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2020 Apr 21. Date of Electronic Publication: 2020 Apr 21.
DOI:	10.1101/2020.04.21.042911
Abstrakt:	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 more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (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 immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (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. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models. Competing Interests: Conflict of interest statement: The authors have declared that no conflict of interest exists.
Databáze:	MEDLINE
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