Development and validation of direct RT-LAMP for SARS-CoV-2

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1. 

   ScreenIT

   Jun 6, 2020

   SciScore for 10.1101/2020.04.29.20075747: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethical approval for use of the archived samples was obtained from the Conjoint Health Research Ethics Board (CHREB) of the University of Calgary (REB20–0402).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Finally, the artificial template containing the targeted sequences of interest was cloned into Sindbis Virus (SV) viral vector system (SINrep5) containing green fluorescent protein (EGFP) and then transfected into BHK-21 cell lines(18),(19).	BHK-21 suggested: ATCC Cat# CRL-6282, RRID:CVC…

   SciScore for 10.1101/2020.04.29.20075747: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethical approval for use of the archived samples was obtained from the Conjoint Health Research Ethics Board (CHREB) of the University of Calgary (REB20–0402).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Finally, the artificial template containing the targeted sequences of interest was cloned into Sindbis Virus (SV) viral vector system (SINrep5) containing green fluorescent protein (EGFP) and then transfected into BHK-21 cell lines(18),(19).	BHK-21 suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   From the multiple sequence alignment, several regions unique to the SARS CoV-2 were identified using our own algorithms developed in collaboration with Illucidx Inc. (Calgary, AB).	AB suggested: RRID:BDSC_203)
   Software and Algorithms
   Sentences	Resources
   LAMP primer design: Genomic sequences (cDNA) of the SARS-CoV-2 were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/) and multiple sequence alignment analysis (https://www.ebi.ac.uk/Tools/msa/clustalo/) was conducted with other related viruses.	https://www.ebi.ac.uk/Tools/msa/clustalo/ suggested: (Clustal Omega, RRID:SCR_001591)
   In a 25 µL LAMP reaction mixture, 1.6 µM F1P and B1P, 0.8 µM LPF and LPB, 0.2 µM F3 and B3 primer concentrations, 8mM MgSO4, 1.4 mM dNTPs, 8 unit of Bst 2.0 WarmStart® DNA Polymerase and 7.5 unit of Warm Start® reverse transcriptase were used.	LAMP suggested: (LAMP, RRID:SCR_001740)
   In silico analysis of primer combinations to determine cross-reactivity: A blast search alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for primers in set 2 (spike gene) and set 3 (RdRP gene) were performed against a critical list of infectious agents that cause upper respiratory tract infections.	https://blast.ncbi.nlm.nih.gov/Blast.cgi suggested: (TBLASTX, RRID:SCR_011823)
   A nucleotide local alignment using BLASTn with the default parameters was performed against the National Center of Biotechnology Information (NCBI) Nucleotide database.	BLASTn suggested: (BLASTN, RRID:SCR_001598)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Limitations of the study include not testing other sample types such as alternate swabs, nasal washes, oropharyngeal samples, sputum, or stool. This work is ongoing with a special emphasis on swab-free testing. Also formal SARS-CoV-2 viral titers were not calculated in the limit of detection studies. Nevertheless, LAMP presents a much needed alternative approach to SARS-CoV-2 diagnostic testing that is available for deployment immediately in a LDT format as it relies on other key reagents that do not cannibalize RT-PCR reagents. Ultimately, the aim is to port LAMP chemistry on a stand-alone microfluidic device POCT to be deployed in the community, either at ports of entry, homes, pharmacies, or resource-limited settings.

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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:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • 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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2. 

   Version published to 10.1101/2020.04.29.20075747v2 on medRxiv

   May 7, 2020
3. 

   Version published to 10.1101/2020.04.29.20075747v1 on medRxiv

   May 4, 2020