Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The SARS-CoV-2 genome was targeted by three sets of primers and probes specific for ORF1ab, ORF3a, and N genes. Red half arrows represented forward and reverse primers. Green half arrow represented probes. The figure was created with BioRender.com.

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques:

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The performance of the real-time RT-PCR assay in the adjusted setting compared with the initial setting (the standard protocol). The mean ± SEM cycle threshold (Ct) values from an experiment with triplicate amplification were compared between two settings in each SARS-CoV-2 gene target (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd). An unpaired t- test was used for comparison analysis. The significant difference was when p < 0.05.

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Amplification, Control, Comparison

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The performance of the adjusted real-time RT-PCR assay ( A ) The RT time was varied from 5 to 4 min, 2 min, and 30 s. The comparison of mean ± SEM Ct values of SARS-CoV-2 gene targets (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd) was analyzed among the different RT time settings using the reference materials; ( B ) Comparing the mean ± SEM Ct values of SARS-CoV-2 gene targets in clinical samples at RT time between 4 min and 30 s. The significant level was calculated using an unpaired t -test and one-way ANOVA with Turkey’s multiple comparison test. The asterisk indicated the statistically significant difference. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Comparison, Control

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The performance of the real-time RT-PCR assay. ( A ) when varying initial denaturation time from 1 min to 30 and 2 s; ( B ) when cycling denaturation and annealing/extension time was compared between 2–4 s and 1–1 s; ( C ) when varying annealing/extension temperatures from 60 to 65 °C; ( D ) when varying denaturation temperatures from 82 to 85, 88, 90, and 92 °C. The comparison of mean ± SEM Ct values of SARS-CoV-2 gene targets (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd) was analyzed among the different conditions. The significant difference was calculated using an unpaired t -test and one-way ANOVA with Turkey’s multiple comparison test. The asterisk indicated the statistically significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Comparison, Control

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The sensitivity of the rapid real-time RT-PCR assay (ƩS COVID-19). The sensitivity of ƩS COVID-19 was determined using the reference SARS-CoV-2 RNA ranging from 10 6 to 1 copies/reaction. Forty cycles of amplification were done in 20 replicates for each concentration of the reference RNA. The amplification plots and the standard curve of the ( A ) ORF1ab, ( B ) ORF3a, and ( C ) N genes of the SARS-CoV-2. The error bar represented the mean ± SEM.

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Amplification, Concentration Assay

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The specificity of the rapid real-time RT-PCR assay (ƩS COVID-19). The specificity of ƩS COVID-19 was determined using 22 respiratory samples positive for other 16 common respiratory viruses (C1-C22) detected by either BioFire Respiratory 2.1 Panel or QIAstat-Dx Respiratory SARS-CoV-2 Panel. The amplification plots of the ( A ) ORF1ab, ( B ) ORF3a, ( C ) N genes of SARS-CoV-2, and ( D ) the internal control (ASBVd).

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Amplification, Control

Journal: Scientific Reports

Article Title: ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

doi: 10.1038/s41598-024-71705-8

Figure Lengend Snippet: The primers and probes used in the rapid real-time RT-PCR assay (ƩS COVID-19).

Article Snippet: Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.

Techniques: Quantitative RT-PCR, Amplification

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – C ) hTERT pre-adipocytes and differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 1). ( A , B ) Relative expression of viral ( A ) genomic RNA (nucleocapsid, *** p = 0.0002, ** p = 0.0018) and ( B ) sub-genomic RNA (ORF1ab, *** p = 0.0003, * p = 0.0263), normalized to β-actin. ( C ) Viral loads measured from supernatant using plaque assay. (**** p < 0.0001, ** p = 0.0024). Data are pooled from two independent experiments ( n = 8, biological replicates). Statistical analysis was performed using two-way ANOVA. ( D ) IL-6 levels in the supernatant of differentiated adipocytes with or without viral infection (MOI = 1), measured by ELISA. Data are pooled from three independent experiments ( n = 14, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( E – G ) Adipocytes were infected at 4, 9, 12, 16, and 20 days post-differentiation (MOI = 1), with measurements taken 48 h post-infection. ( E ) Viral loads measured by plaque assay; data represent two independent experiments ( n = 4, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( F ) Western blot of viral nucleocapsid, FABP4, and β-actin proteins levels in cell lysates. ( G ) Quantification of nucleocapsid and FABP4 band intensities normalized to β-actin, representative of two independent experiments ( n = 4, biological replicates, **** p < 0.0001, *** p = 0.0001 and 0.0006). Statistical analysis was performed using two-way ANOVA. ( H , I ) Percent area of lipid droplets in infected cells and neighboring cells, quantified by fluorescence neutral lipid staining (Bodipy). Infected cells identified by ( H ) nucleocapsid-positive staining and ( I ) dsRNA-positive staining. Data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001, *** p = 0.0001); statistical analysis was performed using a standard t-test. ( J ) Representative confocal images of infected differentiated adipocytes (MOI = 1), stained for dsRNA (red), lipid droplets (yellow), and nucleus (DAPI, blue). Scale bar = 50 μm; magnified regions = 10 μm ( n = 3). ( K ) Percent lipid droplet area relative to dsRNA-positive area per cell. Pearson correlation coefficient indicated as r. ( L , M ) Representative confocal images of control and infected adipocytes at 8, 12, 24, and 48 h post-infection, stained for dsRNA (red), lipid droplets (yellow), calnexin (blue), and FABP4 (gray). ( L ) Merged image of all stains (Scale bar = 50 μm); insets highlight overlap of FABP4, dsRNA, and calnexin (Scale bar = 5 μm). ( M ) Signal overlap between dsRNA, FABP4, and calnexin, and between FABP4 and calnexin individually (Scale bar = 5 μm). ( N – P ) Colocalization of target signals over time, represented as Pearson correlation R. Cells infected with MOI = 3 at 8 and 12 hpi; data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001). For 24 and 48 hpi, MOI = 1 was used ( n = 3) biological replicates. Statistical analysis was performed using one-way ANOVA. Data shown as mean ± s.e.m. .

Article Snippet: SARS-CoV2 ORF1ab , ThermoFisher Scientific , Vi07921935_s1.

Techniques: Infection, Expressing, Plaque Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – D ) Pre-adipocytes and differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 0.1). ( A , B ) Relative expression of viral ( A ) genomic RNA (nucleocapsid, **** p < 0.0001, *** p = 0.0003 and 0.0004) and ( B ) sub-genomic RNA (ORF1ab, **** p < 0.0001), normalized to β-actin. ( C ) Viral loads measured from supernatant using plaque assay (**** p < 0.0001,*** p = 0.0001). Data are pooled from two independent experiments ( n = 8, biological replicates). Statistical analysis was performed using two-way ANOVA. ( D ) IL-6 measured by ELISA in the supernatant of differentiated adipocytes with or without virus infection (MOI = 0.1). Data are pooled from two independent experiments ( n = 8, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( E ) Western blots of SARS-CoV-2 nucleocapsid, FABP4, β-actin protein levels, and total protein (Ponceau S staining) in cell lysates of differentiated adipocytes infected with SARS-CoV-2 (MOI = 0.1 or MOI = 1). ( F ) Quantification of FABP4 band intensity normalized to total protein, representative of two independent experiments ( n = 4, biological replicates). ( G ) FABP4 gene expression relative to β-actin, pooled from two independent experiments ( n = 8, biological replicates). ( H ) FABP4 secretion in the supernatant within 1-hour incubation at the indicated time points following infection. Fold change is calculated relative to uninfected samples. Data are representative of two independent experiments ( n = 6, biological replicates, * p = 0.0336). Statistical analysis was performed using two-way ANOVA. ( I , J ) Representative confocal images of infected adipocytes stained with nucleocapsid (red), FABP4 (green), and lipid droplets (blue). ( I ) Low magnification and ( J ) high magnification images of the same samples (Scale bars = 50 μm, magnified regions = 10 μm) ( n = 3, biological replicates). ( K ) Percentage lipid droplet area relative to nucleocapsid-positive area per cell in infected differentiated adipocytes. Pearson correlation coefficient is indicated as r. Data are shown as the mean ± s.e.m. .

Article Snippet: SARS-CoV2 ORF1ab , ThermoFisher Scientific , Vi07921935_s1.

Techniques: Infection, Expressing, Plaque Assay, Enzyme-linked Immunosorbent Assay, Virus, Western Blot, Staining, Gene Expression, Incubation

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: SARS-CoV2 ORF1ab , ThermoFisher Scientific , Vi07921935_s1.

Techniques: Knock-Out, Sequencing, Recombinant, shRNA, Staining, Electron Microscopy, Plasmid Preparation, Software, Isolation, cDNA Synthesis, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: The primers, reporter molecule and gRNAs used in this study.

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques:

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: Optimal reaction conditions of the COVID-19 MCTOP assay. Error bars represent the means ± standard error of means (SEM) from three replicates. (A) Optimal reaction temperature for ORF1ab gene detection. The reactions were performed under temperatures ranging from 62°C to 66°C at 1°C intervals. (B) Optimal primer concentration for ORF1ab gene detection. Serial volumes of MCDA primer premixture (2.2 μL and 1.1 to 2.3 μL with 0.2 μL intervals) were used to prepare the reaction mixtures. Primer concentrations for serial volumes were as follows. 1.1 μL (0.2 μM each of F1 and F2, 0.4 μM each of C1, C2, R1, R2, D1 and D2, 0.8 μM each of CP1 and CP2), 1.3 μL (0.24 μM each of F1 and F2, 0.47 μM each of C1, C2, R1, R2, D1 and D2, 0.95 μM each of CP1 and CP2), 1.5 μL (0.27 μM each of F1 and F2, 0.55 μM each of C1, C2, R1, R2, D1 and D2, 1.09 μM each of CP1 and CP2), 1.7 μL (0.31 μM each of F1 and F2, 0.62 μM each of C1, C2, R1, R2, D1 and D2, 1.24 μM each of CP1 and CP2), 1.9 μL (0.35 μM each of F1 and F2, 0.69 μM each of C1, C2, R1, R2, D1 and D2, 1.38 μM each of CP1 and CP2), 2.1 μL (0.38 μM each of F1 and F2, 0.76 μM each of C1, C2, R1, R2, D1 and D2, 1.53 μM each of CP1 and CP2), 2.2 μL (0.4 μM each of F1 and F2, 0.8 μM each of C1, C2, R1, R2, D1 and D2, 1.6 μM each of CP1 and CP2), 2.3 μL (0.42 μM each of F1 and F2, 0.84 μM each of C1, C2, R1, R2, D1 and D2, 1.67 μM each of CP1 and CP2). (C) Optimal reaction temperature for N gene detection. The reactions were performed under temperatures ranging from 62°C to 66°C at 1°C intervals. (D) Optimal primer concentration for N gene detection. Serial volumes of MCDA primer premixture (2.0 μL and 1.1 to 2.1 μL with 0.2 μL intervals) were used to prepare the reaction mixtures. Primer concentrations for serial volumes were as follows. 1.1 μL (0.22 μM each of F1 and F2, 0.44 μM each of C1, C2, R1, R2, D1 and D2, 0.88 μM each of CP1 and CP2), 1.3 μL (0.26 μM each of F1 and F2, 0.52 μM each of C1, C2, R1, R2, D1 and D2, 1.04 μM each of CP1 and CP2), 1.5 μL (0.3 μM each of F1 and F2, 0.6 μM each of C1, C2, R1, R2, D1 and D2, 1.2 μM each of CP1 and CP2), 1.7 μL (0.34 μM each of F1 and F2, 0.68 μM each of C1, C2, R1, R2, D1 and D2, 1.36 μM each of CP1 and CP2), 1.9 μL (0.38 μM each of F1 and F2, 0.76 μM each of C1, C2, R1, R2, D1 and D2, 1.52 μM each of CP1 and CP2), 2.0 μL (0.4 μM each of F1 and F2, 0.8 μM each of C1, C2, R1, R2, D1 and D2, 1.6 μM each of CP1 and CP2), 2.1 μL (0.42 μM each of F1 and F2, 0.84 μM each of C1, C2, R1, R2, D1 and D2, 1.68 μM each of CP1 and CP2). Ultrapure water served as negative control (NC).

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Concentration Assay, Negative Control

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: Sensitivity and specificity analysis of COVID-19 MCTOP assay. (A,C) COVID-19 MCTOP results were reported by real-time fluorescence (A) for ORF1ab-MCTOP result, and (C) for N-MCTOP result. Error bars represent the means ± standard error of means (SEM) from three replicates. (B,D) COVID-19 results were reported by lateral flow biosensor (B) for ORF1ab-MCTOP result, and (D) for NP-MCTOP result. Signals (A) /Biosensors (B) 1–8 represented the results of corresponding ORF1ab-plasmid levels of SARS-CoV-2 from 1 × 10 4 to 1 × 10 0 copies, and 1 × 10 4 copies ORF1ab-plasmid of SARS-CoV and MERS-CoV, respectively, and ultrapure water served as negative control (NC). Signals (C) /Biosensors (D) 1–8 represented the results of corresponding N-plasmid levels of SARS-CoV-2 from 1 × 10 4 to 1 × 10 0 copies, and 1 × 10 4 copies N-plasmid of SARS-CoV and MERS-CoV respectively, and ultrapure water served as negative control (NC). Control line (CL), Test line (TL).

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Fluorescence, Plasmid Preparation, Negative Control, Control

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: The effects of reaction inhibitors on COVID-19 MCTOP assay. 1 × 10 4 copies of SARS-CoV-2 plasmid were used in the tolerance test. (A) For ORF1ab-MCTOP result, (B) for N-MCTOP result. Ultrapure water served as negative control (NC). Error bars represent the means ± standard error of means (SEM) from at three replicates.

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Plasmid Preparation, Negative Control