Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Purified SARS-CoV-2 RdRp proteins expressed in baculovirus-infected insect cells ( Spodoptera frugiperda , Sf) analysed by SDS–PAGE and Coomassie staining. 7H8: nsp7-His 6 -nsp8, nsp12-HF: nsp12-His 6 -3xFlag, nsp12-F/7H8: nsp12-3xFlag/nsp7-His 6 -nsp8, nsp12-HF/7L8: nsp12-His 6 -3xFlag/nsp7-GGSGGS-nsp8, nsp12-HF/7/8: nsp12-His 6 -3xFlag/nsp7/nsp8. ( B ) Bacterially expressed and purified SARS-CoV-2 nsp7, nsp8 and nsp12 proteins analysed by SDS–PAGE and Coomassie staining. The proteins were expressed as 14His-SUMO fusion proteins in E. coli . 14His-SUMO was removed by a SUMO-specific protease during purification generating native N-termini. ( C ) Gel-based primer-extension assay to test RNA-dependent RNA synthesis using the RdRp preparation Sf nsp12-F/7H8. The substrate consists of a 10 nt RNA primer annealed to the 3′ end of a 35 nt RNA template. The 5′ end of the template strand is labelled with a Cy3 fluorophore. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. Formation of duplex RNA by RdRp was observed over time. Controls: a preformed Cy3-labelled dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Schematic diagram illustrating the FRET-based RdRp strand displacement assay. The RNA substrate is composed of a Cy3 fluorophore-containing template strand, an annealed primer and an annealed quencher strand with a 5′ flap. RdRp activity synthesises RNA by extending the primer strand and displaces the quencher strand. The displaced quencher strand can no longer anneal to fully synthesised duplex RNA leading to an increase in Cy3 fluorescent signal. ( E ) FRET-based strand displacement assay using the indicated concentrations of Sf nsp12-F/7H8.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Purification, Infection, SDS Page, Staining, Primer Extension Assay, Clear Native PAGE, Fluorescence, Activity Assay

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Logistics of the screen. A custom chemical library consisting of over 5000 compounds was screened against RdRp activity using the FRET-based strand displacement assay in a 384-well format. RdRp was prepared by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 in a 1 : 3 ratio for 30 min at room temperature. RdRp was dispensed into compound-containing 384-well plates and incubated for 10 min. Reactions were started by the addition of a substrate mix and florescence monitored in 90 s intervals. ( B,C ) Results of the HTS screen performed at 1.25 µM ( B ) and 3.75 µM ( C ) compound concentration. The normalised reaction velocity plotted against the compound number is shown. Validated hits are shown in red. ( D ) Kinetic curves with >15% reduction in reaction velocity or >10% reduction in fluorescent signal at the endpoint were inspected manually. As example, kinetic data for compound GSK-650394 is shown (red curve, data from surrounding wells in black). ( E ) Summary of the HTS hit selection strategy. From over 5000 compounds tested in the screen, 64 were considered primary hits after manual inspection of HTS reactions, which showed a reduction in reaction velocity below 85%, or a reduction in endpoint signal below 90%. Out of these, 46 primary hits were eliminated as they likely represent nonspecific modes of enzymatic inhibition such as colloidal aggregation or interference with the substrate structure. As part of this analysis promiscuous compounds that were identified as hits in other SARS-CoV-2 HTS [ , ] were removed with the exception of five suramin and suramin-like compounds, which were also identified in the SARS-CoV-2 nsp13 helicase HTS (Zeng et al. ). In vitro validation of the effect of suramin and suramin-like compounds on the activity of SARS-CoV-2 helicase and SARS-CoV-2 RdRp can be found in Zeng et al. . A total of 18 compounds (including five suramin and suramin-like compounds) were selected as hits, of which 14 were included in further in vitro validation in this work.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Activity Assay, Incubation, Concentration Assay, Selection, Inhibition, In Vitro

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Viral inhibition experiments workflow graphical representation. In brief, 24 h after Vero E6 cells were seeded in a 96-well format, they were treated with selected drugs at specified concentrations. Then, cells were infected with a SARS-CoV-2 isolate at a MOI of 0.5 PFU per cell. Twenty-two hours later, cells were fixed and analysed by immunofluorescence staining and imaging. ( B ) SARS-CoV-2 antiviral activity of GSK-650394, C646, BH3I-1, MDK-83190 and cefsulodin. Representative overlaid images of viral N protein immunofluorescence (green) and DNA dye DRAQ7 staining (red). ( C ) Dose–response curve analysis. Viral infection values represent the area of viral plaques visualised by viral nucleocapsid protein staining (green curves) and cell viability was measured in the same experiment as the area of cells stained with the DNA dye DRAQ7 (red curves). Data are plotted as percentage relative to DMSO only control wells (100%). Data represent the mean and standard deviation (SD) of 3 replicates. Areas were calculated using FIJI and EC 50 values were calculated using Prism software.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Inhibition, Infection, Immunofluorescence, Staining, Imaging, Activity Assay, Standard Deviation, Software

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Effective SDS and Tween concentration determination for qPCR and virus inactivation. ( A ) Determination of the minimal Tween 20 and Tween 80 concentration necessary for SDS sequestration to enable amplification of 20,000 copies/µL of RNase P DNA. Ct cycle threshold, ND not detected. ( B ) Fluorescence assay of eGFP-expressing recombinant VSV-SARS-CoV-2 (10 7 pfu/mL) infecting Vero-E6 cells after incubation with 0.1% (w/v) SDS for 15–120 s. Only a sample that was not exposed to SDS showed infectivity. 15 s exposure to 0.1% (w/v) SDS proved sufficient to completely inactivate the virus, likely due to dissolution of its membrane and denaturation of the sample proteins.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Concentration Assay, Amplification, Fluorescence, Expressing, Recombinant, Incubation, Infection

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Comparison of standard qPCR to SDS/Tween and SDS/Tween agarose reactions. ( A ) Comparison of 10 µL qPCR reactions for SARS-CoV-2 N1 gene DNA amplification, containing 3% (v/v) of Tween 80 with (R 2 = 0.9781) and without 0.01% SDS (w/v, final; R 2 = 0.9818) and 1% (w/v) agarose (R 2 = 0.9260). ( B ) Comparison of a 10 µL qPCR reaction for SARS-CoV-2 N1 gene DNA containing 3% (v/v) Tween 80 (R 2 = 0.9908) with a 5 µL qPCR reaction (R 2 = 0.8451) for SARS-CoV-2 N1 DNA detection, containing 3% (v/v) Tween 80, 0.01% SDS (w/v, final) and 1% (w/v) agarose. In all conditions containing SDS, the DNA samples were initially incubated with 0.1% SDS (w/v) in Tris/EDTA buffer to simulate denaturing conditions for clinical samples. Ct cycle threshold.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Amplification, Incubation

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Single-tube sample-to-assay RT-qPCR of SARS-CoV-2 positive-patient RNA sample and inactivated viral particles. ( A ) Comparison of five-fold diluted purified and unpurified SARS-CoV-2 patient RNA samples (nasal swabs stored in VTM + 0.5% [w/v] SDS) in triplicate 10 µL RT-qPCR reactions containing SARS-CoV-2 N1 primers and probe, 3% (v/v) Tween 80, and 0.5% (w/v) agarose. Samples were diluted five-fold to achieve a 0.1% (w/v) SDS working concentration for unpurified samples in VTM. Data are presented as ± SEM. ( B ) Comparison of 5 µL and 10 µL RT-qPCR reactions containing 3% (v/v) Tween 80, and 0.5% (w/v) agarose with a purified SARS-CoV-2 patient RNA sample. Triplicate reactions were performed at each volume, comparing the patient sample with and without spiking into saliva. Reactions were performed with SARS-CoV-2 N1 and beta-actin (ACTB) primer–probe sets. Data are presented as ± SEM. ( C ) RT-qPCR results of SARS-CoV-2 synthetic RNA (n = 11) in water. RT-qPCR was performed for 45 cycles using SARS-CoV-2 N1- and N2-specific primers and probes on a Quantabio Q thermal cycler. Samples were tested blind with concentrations and storage media revealed thereafter (ND = not detected). ( D ) Scatter plot of Ct value (y-axis) versus log[copies] of SARS-CoV-2 viral particles (x-axis) of inactivated SARS-CoV-2 viral particle samples (n = 49) in PBS, saliva, and nasal media. RT-qPCR was performed for 45 cycles with SARS-CoV-2 N1-specific primers and fluorescent probe on a Bio-Rad CFX Connect thermal cycler. Ct cycle threshold.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Quantitative RT-PCR, Purification, Concentration Assay

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: RT-LAMP amplification with SDS and Tween 80. ( A ) Real-time fluorescence of SARS-CoV-2N gene amplification by RT-LAMP was performed at 65 °C for 50 min. Indicated concentrations (copies) of SARS-CoV-2 viral particles were assessed including a no-reverse-transcriptase (NRT) and no-template control (NTC; 0 copies), with all SARS-CoV-2 samples providing a positive result after ~ 30 min. ( B ) Amplification products (1 µL) from the real-time fluorescence RT-LAMP reaction were fractionated using agarose gel electrophoresis. The expected laddering pattern of LAMP amplification products was observed at all SARS-CoV-2 viral particle concentrations and not observed in NRT and NTC controls. The uncropped gel can be found in Supplementary Fig. . ( C ) Fluorescence of RT-LAMP reactions of SARS-CoV-2 viral particles containing 20 µM SYTO 82 observed after 30 and 50 min of incubation at 65 °C. Reactions were analyzed with white, green, and UV (365 nm) handheld LED flashlights at each time point, with readily observable fluorescence of amplification reactions starting from 100 to 10 copies (labeled) after 30 min with each illumination. After 50 min, fluorescence in the 100-copy and 10-copy tubes increased with no observable amplification in the 0-copy control. RFU relative fluorescence units.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Amplification, Fluorescence, Agarose Gel Electrophoresis, Incubation, Labeling

Journal: Oral Diseases

Article Title: The diagnostic accuracy of saliva testing for SARS‐CoV‐2: A systematic review and meta‐analysis

doi: 10.1111/odi.13934

Figure Lengend Snippet: Sensitivity and specificity of saliva tests for detecting SARS‐CoV‐2. Forest plots of individual/pooled sensitivity and specificity of the included studies (CI: confidence interval; Q: Cochran chi‐square test)

Article Snippet: Assay , One‐Step RT‐ddPCR Advanced Kit for Probes (Bio‐Rad) , TaqPath TM COVID‐19 multiplex real‐time RT‐PCR test (Thermo Fisher Scientific) , SuperScript III Platinium One‐Step real time RT‐PCR kit , SARS‐CoV‐2 RNA dependent RNA polymerase/Helicase (RdRp/Hel) real‐time RT‐PCR , Genesis RT‐PCR SARS‐CoV‐2 (Primer Design) kit , Hologic Aptima SARS‐CoV‐2 TMA assay (Hologic Inc.) real‐time RT‐PCR platform , Allplex 2019‐nCoV Assay (100T) (Seegene Inc) , In house RT‐PCR based on CDC assay.

Techniques:

Journal: Oral Diseases

Article Title: The diagnostic accuracy of saliva testing for SARS‐CoV‐2: A systematic review and meta‐analysis

doi: 10.1111/odi.13934

Figure Lengend Snippet: Diagnostic test accuracy of saliva test for detecting SARS‐CoV‐2 (SROC: summary receiver operating characteristic; SENS: sensitivity; SPEC: specificity; AUC: area under the curve; O: observed data; ♦: Summary Oberating Point; ➖: SORC curve; ‐‐‐: 95% confidence contour; …… 95% prediction contour; 1: Aita et al., ; 2: Altawalah et al., ; 3: Binder et al., ; 4: Chen et al., ; 5: Guclu et al., ; 6: Hanson et al., ; 7: Jamal et al., ; 8: Landry et al., ; 9: Moreno‐Contreras et al., ; 10: Pasomsub et al., ; 11: Procop et al., ; 12: Rao et al., ; 13: Sakanashi et al., ; 14: Senok et al., ; 15: Vaz et al., ; 16: Williams et al., ).

Article Snippet: Assay , One‐Step RT‐ddPCR Advanced Kit for Probes (Bio‐Rad) , TaqPath TM COVID‐19 multiplex real‐time RT‐PCR test (Thermo Fisher Scientific) , SuperScript III Platinium One‐Step real time RT‐PCR kit , SARS‐CoV‐2 RNA dependent RNA polymerase/Helicase (RdRp/Hel) real‐time RT‐PCR , Genesis RT‐PCR SARS‐CoV‐2 (Primer Design) kit , Hologic Aptima SARS‐CoV‐2 TMA assay (Hologic Inc.) real‐time RT‐PCR platform , Allplex 2019‐nCoV Assay (100T) (Seegene Inc) , In house RT‐PCR based on CDC assay.

Techniques: Diagnostic Assay

Journal: International Journal of Molecular Sciences

Article Title: The Local Anaesthetic Procaine Prodrugs ProcCluster ® and Procaine Hydrochloride Impair SARS-CoV-2 Replication and Egress In Vitro

doi: 10.3390/ijms241914584

Figure Lengend Snippet: List of all primers used.

Article Snippet: SARS-CoV-2 Spike fw , 5′-CAACTGAAATCTATCAGGCCG-3′ , OriGene #HP234776.

Techniques: Sequencing