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

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1. Version published to 10.1172/jci.insight.139042

   Jun 18, 2020
2. 

   ScreenIT

   Jun 6, 2020

   SciScore for 10.1101/2020.02.07.939389: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All procedures in this study involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (BLL20001).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Animal experiments: For the animal experiments, specific pathogen-free, 6-11-month-old, male and female transgenic hACE2 mice were obtained from the Institute of Laboratory Animal Science, Peking Union Medical College, China.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   96-well plates were coated with Spike 1 …

   SciScore for 10.1101/2020.02.07.939389: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All procedures in this study involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (BLL20001).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Animal experiments: For the animal experiments, specific pathogen-free, 6-11-month-old, male and female transgenic hACE2 mice were obtained from the Institute of Laboratory Animal Science, Peking Union Medical College, China.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   96-well plates were coated with Spike 1 protein of SARS-CoV-2 (0.1μg/100 μL, Sino Biological, 40591-V08H), the tested sera were diluted at 1:100 and added to each well, and 3 multiple wells were set for each sample, and then incubated at 37°C for 30 minutes, followed by the goat anti-mouse secondary antibodies conjugated with HRP (ZB-2305, zhongshan,1:10,000 dilution), and incubated at room temperature for 30 minutes.	anti-mouse suggested: (ZSGB-Bio Cat# ZB-2305, RRID:AB_2747415)
   After blocking in 1% normal goat serum, the sections were incubated with 7D2 monoclonal antibody (laboratory preparation) at 4 °C overnight, followed by HRP-labeled goat anti-mouse IgG secondary antibody (HRP) (Beijing ZSGB Biotechnology, ZDR-5307).	anti-mouse IgG suggested: None
   Alternatively, the sections were stained with MAC2 antibody (Cedarlane Laboratories, CL8942AP), CD3 antibody (Dako, A0452) or CD19 antibody (Cell Signaling Technology, 3574) at 4°C overnight.	MAC2 suggested: (CEDARLANE Cat# CL8942AP, RRID:AB_10060357) CD3 suggested: (Agilent Cat# A0452, RRID:AB_2335677) CD19 suggested: (Cell Signaling Technology Cat# 3574, RRID:AB_2275523)
   Subsequently, the sections were goat anti-rat IgG secondary antibody (HRP) (Beijing ZSGB Biotechnology, PV9004), goat anti-rabbit IgG secondary antibody (HRP) (Beijing ZSGB Biotechnology, PV9001) for 60 min, and visualized by incubation with 3,30-diaminobenzidine tetrahydrochloride (DAB).	goat anti-rat IgG secondary antibody (HRP) (Beijing ZSGB Biotechnology, PV9004) suggested: None anti-rat IgG suggested: (ZSGB-Bio Cat# PV-9004, RRID:AB_2868453) goat suggested: (ZSGB-Bio Cat# PV-9001, RRID:AB_2868452) IgG secondary antibody (HRP) (Beijing ZSGB Biotechnology, PV9001) for 60 min, suggested: None
   Anti-S protein antibody (mouse monoclonal 7D2, laboratory preparation, 1:200) and anti-hACE2 antibody (rabbit polyclonal, ab15348, Abcam1:200) were used as the primary antibody.	anti-hACE2 suggested: None
   The sections were washed with PBS and incubated with secondary antibodies conjugated with FITC (goat anti-mouse, ZF-0312, Beijing ZSGB Biotechnology, 1:200) and TRITC (goat anti rabbit, ZF-0317, Beijing ZSGB Biotechnology, 1:200), dried at room temperature and observed via fluorescence microscopy.	anti-mouse, ZF-0312 suggested: (ZSGB-Bio Cat# ZF-0312, RRID:AB_2716306) anti rabbit, ZF-0317 suggested: None
   For the expression of hACE2, the sections from WT mice stained with anti-ACE2 antibody were used as the negative control, and the stable cell line expressing hACE2 was used as the positive control.	anti-ACE2 suggested: None
   For the viral antigen, the sections from ACE2-Mock mice incubated with anti-S protein antibody were used as the negative control.	anti-S protein suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Seed SARS-CoV-2 stocks and virus isolation studies were performed in Vero cells, which are maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin, and incubated at 37°C, 5% CO2.	Vero suggested: None
   Transmission Electron Microscopy: Supernatant from Vero E6 cell cultures that showed cytopathic effects was collected, inactivated with 2% paraformaldehyde for at least 2 hours, and ultracentrifuged to sediment virus particles.	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   ELISA method: The specific IgG against SARS-CoV from ACE2-HB-01 mice and WT-HB-01 mice were determined by enzyme-linked immunosorbent assay (ELISA).	ACE2-HB-01 suggested: None WT-HB-01 suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical analysis: All data were analyzed with GraphPad Prism 8.0 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2020.04.21.042911v1 on bioRxiv

   Apr 22, 2020