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Supplementary Figures and Tables: Isothermal amplification using sequence-specific fluorescence detection of SARS-CoV-2 and variants in nasal swabs

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Supplemental   Figure 1. FQ-LAMP LOD for WA1 - Reaction Tube Images

WA1 Target Log Dilution in SPS. Culture   supernatants were log-serially diluted from 106 /ml to 10 pfu/ml   in the SPS buffer and analyzed by FQ-LAMP. B. WA1   Target Two-fold Dilution in SPS.  WA1-infected Vero cell culture supernatant two-fold   diluted in SPS buffer from 1,000 to 32 PFU/ml was processed with the SPS   protocol and analyzed by FQ-LAMP C. No   Template Control. FQ-LAMP assay   using identically processed samples, but lacking WA1 with deionized water   added to the SPS buffer served as the no-template control (NTC). Images were acquired using a mobile phone   camera with no filters and excitation using a handheld UV lamp (302nm).

Supplemental Figure 2. FQ-LAMP   WA1 Target Multiples to Confirm LOD – Reaction Tube Images.

 FQ-LAMP assay targeting WA1 from infected Vero   cells. Culture supernatants were spiked into pooled CoV2-negative nasal swab   samples at: A. 2,000; B. 1,000;   and C. 500 PFU/ml representing 2X,   1X, and 0.5X LOD target concentration, respectively. All samples were   processed with SPS buffer as described above using n=20 replicates for each   target dilution. D. Negative control assays (no spike), n=20   replicates used identically processed nasla swab sample but without WA1. Images acquired using a mobile phone camera   with no optical filters under excitation with a laboratory handheld UV lamp   (302nm). 1,000 PFU/ml WA1 target   concentration was the highest dilution displaying at least 19/20 replicates   with positive signal and was determined to be the assay LOD. 

Supplemental Figure 3. FQ-LAMP   CoV2 Variants in Nasal Swab Sample (2,000 PFU/ml) Reaction Tube Images

FQ-LAMP targeting CoV2   variant viruses:  A. Delta (B.1.617.2), B.  Alpha (B.1.1.7), and C. Omicron (B.1.1.529) from infected Vero cell cultures. Culture supernatants were spiked into   pooled CoV2-negative nasal swab sample at 2,000 PFU/ml (2X LOD for WA1) and   processed with SPS buffer as described above using n=20 replicates for each   target virus. Images acquired using a   mobile phone camera with no filters under excitation with a standard   laboratory handheld UV lamp (302nm). CoV2 variant viruses are easily detectable by FQ-LAMP in nasal swab   sample background at 2X LOD, and all the heterologous virus targets, even at   very high concentrations, failed to produce fluorescence signals over the   background in the FQ-LAMP assay (images not shown). 

Supplemental Table 1. FQ-LAMP targeting WA1 from infected Vero cells.   Culture supernatants were spiked into pooled CoV2-negative nasal swab samples   at 2,000, 1,000; and 500 PFU/ml representing 2X, 1X, and 0.5X LOD target   concentration, respectively, and processed with SPS buffer as described using   n=20 replicates for each target dilution (three representative samples are   pictured).  1,000 PFU/ml WA1 target   concentration was the highest dilution displaying at least 19/20 replicates   with a positive signal in the LOD. FQ-LAMP assay targeting CoV2 variant viruses Delta (B.1.617.2), Alpha   (B.1.1.7), and Omicron (B.1.1.529) from infected Vero cell culture. Culture supernatants were spiked into   pooled CoV2-negative nasal swab sample at 2,000 PFU/ml (2X LOD for WA1) and   processed with SPS buffer as described above using n=20 replicates for each   target virus, and FQ-LAMP targeting three human coronavirus strains i.e. OC43   (GenBank: AY585228.1), 229E (GenBank: AF304460.1), and NL63 (GenBank:   AY567487.2) from infected Vero cells, human respiratory syncytial virus   strain A2, GenBank: KT992094.1) from infected Vero cells, and influenza A   virus A/Guangdong-Maonan/SWL1536/2019 (H1N1, GISAID: EPI_ISL_419003) from   infected embryonated hen eggs. Culture supernatants, or egg lysate for IAV,   were spiked into pooled SARS-CoV-2 negative nasal swab sample at 105 PFU/ml   and processed with SPS buffer as described using n=20 replicates for each   target virus, and negative control assays (no spike), n=20 replicates   consisting of identically processed nasal swab sample, but without CoV2   variant virus. CoV2 variant viruses are easily detectable by FQ-LAMP in nasal   swab sample background at 2X LOD, and all the heterologous virus targets,   even at very high concentrations, did not produce a fluorescence signal over the background in   the FQ-LAMP assay.  

Supplemental Table 2. CoV2 N   gene-based FQ-LAMP primers and signal oligos were created using free Primer   Explorer V software. Numbering   according to CoV2 Wuhan-Hu-1 (GenBank: MN908947.3). 

Funding

R.A.T. is on the scientific advisory board of Trellis Biosciences. The authors would like to thank the Georgia Research Foundation for supporting the studies, and Trellis Biosciences for their support in LAMP development. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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