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: ACS Sensors

Article Title: Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

doi: 10.1021/acssensors.1c00702

Figure Lengend Snippet: Design of the assay and its operation. (a) Photographs showing components of the developed POC toolkit. The top cover is a plastic cover for the thermal isolation of the running assay. The internal heat module (IH module) consists of several reservoirs filled with CaO powder, water, and wax. A cellulose paper strip is placed between the CaO and water reservoirs to carry the water to the CaO reservoir. The extraction and detection module (ED module) is a multilayered paper platform. The flow paths for manipulating different reagents are defined by imprinting on the paper with a water-insoluble ink (brown). Timers were imprinted (green) on the flow paths to introduce controlled amounts of delay to the flow of different reagents. (b) Exploded image of the ED module showing individual layers. Two paper layers imprinted with features were laminated in between polymer tapes and were coupled through the detection spots punched out of filter paper pretreated with chemicals for RNA extraction. The primer mixtures specifically targeting SARS-CoV-2 and influenza A and B viruses were dried in front of the detection spots to be carried by the RT-LAMP buffer flow on the second paper layer. (c) Time-lapse images of the ED module showing its capability to coordinate the delivery of four dye solutions simultaneously loaded into the module. The dye solutions were used instead of the actual sample and reagents for visual investigation. The images show the state of the module at selected timepoints: the delivery of the saliva sample (yellow), proteinase K (green), DI water (red), and RT-LAMP buffer (blue) to the detection spots. (d) Schematic showing the procedure to operate the developed assay. The presence of SARS-CoV-2 and influenza A and B viruses in the processed sample can be visually identified by the color changes in the corresponding detection spot.

Article Snippet: The SARS-CoV-2 PC template (N gene, 1000 copies/μL, 1 μL) and SARS-CoV-2 primer (New England BioLabs, MA) were predried on the PC spot.

Techniques: Isolation, Stripping Membranes, Introduce, RNA Extraction

Journal: ACS Sensors

Article Title: Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

doi: 10.1021/acssensors.1c00702

Figure Lengend Snippet: Characterization and optimization of viral RNA extraction. (a) Amplification plots from the real-time RT-LAMP of the extracted SARS-CoV-2 on five different filter papers: cellulose and nitrocellulose paper as controls and three commercially available nucleic acid sampling papers. No amplifications were observed on the cellulose and nitrocellulose papers. Amplification occurred on the FTA, FTA Elute, and RNA Sound cards with different T t values. For each amplification, measurements were performed in pairs to ensure against artifacts. (b) Measured mean T t value differences (Δ T t ) for different papers tested. The error bars represent the standard deviation ( n = 3). (c) Measured amplification curve when the SARS-CoV-2 RNA was autonomously extracted by an ED module equipped with detection spots made out of the FTA Elute card and was subsequently subjected to real-time RT-LAMP outside of the device.

Article Snippet: The SARS-CoV-2 PC template (N gene, 1000 copies/μL, 1 μL) and SARS-CoV-2 primer (New England BioLabs, MA) were predried on the PC spot.

Techniques: RNA Extraction, Amplification, Sampling, Standard Deviation

Journal: ACS Sensors

Article Title: Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

doi: 10.1021/acssensors.1c00702

Figure Lengend Snippet: Autonomous RNA amplification and colorimetric detection. (a) Schematic showing the functions of individual components of the IH module. The exothermic reaction of CaO with water produces heat, and the melting of the wax regulates the temperature of the device to the desired range for the RNA amplification. (b) Measured temperature of the device with (red) and without (black) the wax following the onset of the exothermic reaction. The melting of the wax regulates the device temperature at ∼68–72 °C and prevented a temperature spike that would inactivate the Bst polymerase. (c) Measured device temperature loaded with different amounts of CaO. CaO (5 g) could maintain an isothermal environment at ∼68 °C for >90 min. (d) Images of the detection spots taken at different timepoints showing the changes in the spot colors as the reaction progresses. The images show the color on PC and SARS-CoV-2 spots changing from pink to yellow after 40 min, while the color on NC spot remains pink for the whole tested duration. (e) Measured color intensity of the PC, NC, and SARS-CoV-2 spots as functions of the reaction time. Error bars in all panels represent the standard deviation ( n = 3).

Article Snippet: The SARS-CoV-2 PC template (N gene, 1000 copies/μL, 1 μL) and SARS-CoV-2 primer (New England BioLabs, MA) were predried on the PC spot.

Techniques: Amplification, Standard Deviation

Journal: ACS Sensors

Article Title: Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

doi: 10.1021/acssensors.1c00702

Figure Lengend Snippet: Multiplex detection of SARS-CoV-2 and influenza A and B viruses. (a) Assay results corresponding to the saliva samples spiked with different copy numbers of SARS-CoV-2, influenza A, and influenza B viruses. Among the three central detection spots, the left one tests for the presence of SARS-CoV-2, the middle one tests for influenza A, and the right spot tests for the influenza B virus. The change in the color of a spot indicates a positive result for the targeted virus. (b) Plots below the images show the measured color intensities for each test as functions of virus copy number. Error bars represent the standard deviation ( n = 3).

Article Snippet: The SARS-CoV-2 PC template (N gene, 1000 copies/μL, 1 μL) and SARS-CoV-2 primer (New England BioLabs, MA) were predried on the PC spot.

Techniques: Multiplex Assay, Standard Deviation

Journal: ACS Sensors

Article Title: Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

doi: 10.1021/acssensors.1c00702

Figure Lengend Snippet: Application of the assay on COVID-19 patient samples. (a) Assay results corresponding to the saliva samples from three different COVID-19 patients (left) and healthy donors as controls (right). The leftmost and rightmost spots on the assay are PCs and NCs, respectively. Among the three central detection spots, the left one tests for the presence of SARS-CoV-2, the middle one tests for influenza A, and the right spot tests for the influenza B virus. The change in the color of the left spot for patient samples indicates a positive result for the SARS-CoV-2. (b) Plots below the images show the corresponding normalized color intensities for each spot for all tested saliva samples from COVID-19 patients (left) and healthy donors (right).

Article Snippet: The SARS-CoV-2 PC template (N gene, 1000 copies/μL, 1 μL) and SARS-CoV-2 primer (New England BioLabs, MA) were predried on the PC spot.

Techniques: