Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes
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SciScore for 10.1101/2020.12.26.20248878: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Non-patient Test Samples and transport media: Normal human saliva was purchased from BioIVT from 5 males and 5 females with ages ranging from 27-42 yrs old, and a mix of races including Asian, Black, Caucasian, and Hispanic. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus sample processing and yields: Human coronavirus 229E (HCov229E) was obtained from ATCC (VR-740) and propagated in MRC-5 cells cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum … SciScore for 10.1101/2020.12.26.20248878: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Non-patient Test Samples and transport media: Normal human saliva was purchased from BioIVT from 5 males and 5 females with ages ranging from 27-42 yrs old, and a mix of races including Asian, Black, Caucasian, and Hispanic. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus sample processing and yields: Human coronavirus 229E (HCov229E) was obtained from ATCC (VR-740) and propagated in MRC-5 cells cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic solution (Gibco) at 37 °C, 5% CO2 incubator. MRC-5suggested: NoneFor the RPP30 RNA standard, cellular RNA from HEK293T cells was extracted, cDNA was synthesized, PCR was performed with T7 promoter containing primer sets and the in vitro transcribed RNA was prepared as described above. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Each HEK293 cell was determined to contain on average 9.5 copies of detectable RPP30 RNA. HEK293suggested: NoneSoftware and Algorithms Sentences Resources We selected 43,849 sequences that had an identifiable N gene using the search string ATGTCTGATAATGGACCCCA from RefSeq NC_045512.2 for the SARS-CoV-2 N gene. RefSeqsuggested: (RefSeq, RRID:SCR_003496)Sequence homology between the DW LAMP primer set and SARS-CoV ZJ01 was determined using the “Map Primers” function in Geneious Prime, with NCBI accession AY297028 as the reference and DW LAMP primers truncated to remove the loop regions as the query. NCBIsuggested: (NCBI, RRID:SCR_006472)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).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:An additional caveat here is that HCoV-229E may not behave as SARS-CoV-2 in direct assays, as others have reported 97% sensitivity with a similar heat treatment14. Regardless, our data is consistent with others that direct assays and heat inactivation both support successful RT-LAMP assays, with less sensitivity relative to qRT-PCR using purified viral RNA. We consider this loss of sensitivity a reasonable trade-off in circumventing the disadvantages of RNA purification as a first step. Direct SARS-CoV-2 LAMP assays have been previously tested with varying degrees of success16–20. Regarding sensitivity, we have demonstrated a 95% LoD of 22.6 copies per assay (11.3 copies/µL) and a lower limit of about 3 copies per assay (1.5 copies/µL) in normal saline (Fig 4a). This analytical sensitivity is essentially the same as the CDC’s 2019-nCoV Real-Time RT-PCR Diagnostic Panel with an LoD of 10 copies/µL 21(https://www.fda.gov/media/134922/download). Vogels and colleagues22 surveyed the performance of multiple qRT-PCR SARS-CoV-2 primer sets used in testing worldwide and determined that the best of these detected 1 copy/µL at 25% frequency and 10 copies/µL at 25-50% frequency. Although their assay conditions were less than optimal, the jLAMP assay described here performed similarly at these very low copy numbers (Figure 5a). It should also be noted that the LoD values are analytical sensitivity measurements, and not a measure of clinical performance. In clinical samples, the jLAMP ass...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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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