Journal: Frontiers in immunology

Article Title: Graphene quantum dots induce cascadic apoptosis via interaction with proteins associated with anti-oxidation after endocytosis by Trypanosoma brucei .

doi: 10.3389/fimmu.2022.1022050

Figure Lengend Snippet: FIGURE 3 Cytotoxicological effects graphene quantum dots (GQDs) on Trypanosoma brucei 12 and 24 h post-exposure. (A) Quantitative analysis of T. brucei intracellular reactive oxygen species (ROS) values based on the results in Supplementary Figure S7. The intracellular ROS levels increased with prolonged exposure and increased doses. (B) Alterations of the MMP (DYm) in T. brucei following treatment with GQDs. The levels of DYm decreased with increased doses and duration of exposure. A JC-10 fluorescent probe was applied for the detection using flow cytometry (Supplementary Figure S8). (C) Increased cytoplasmic Ca2+ concentration of T. brucei following treatment with GQDs for 12 and 24 h, demonstrated by flow cytometry. Changes in the intracytoplasmic Ca2+ concentration captured by confocal microscopy, which can be seen in Supplementary Figure S9. (D) Intracellular ATP was significantly decreased in T. brucei after exposure to GQDs. (E) Exposure to GQDs resulted in the developmental arrest of T. brucei. The proportions of cell cycle dispersion are shown in graphs, predicated in the flow cytometry data in Supplementary Figure S10. (F) Flow cytometry analysis of the increased DNA fragmentation in T. brucei after treatment with varying concentrations of GQDs for 12 and 24 h using TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. T. brucei parasites that were not exposed to GQDs served controls. All values are the mean ± SD of triplicates. Student’s t-test was used to obtain p- values. *p < 0.05, **p < 0.01, ***p < 0.001 (compared to the control).

Article Snippet: The annexin V-FITC Apoptosis Detection Kit (C1062L), Cell Cycle and Apoptosis Analysis Kit (C1052), Cell Counting Kit-8 (C0038), and one-step TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) Apoptosis Assay Kit (C1088) were provided by Beyotime Biotechnology (Suzhou, China).

Techniques: Cytometry, Concentration Assay, Confocal Microscopy, Dispersion, Flow Cytometry, TUNEL Assay, Staining, Control

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: (A) NP swab diluents from 2 confirmed COVID-19 patients were pooled, and using the 2019-nCoV_N3 primer/probe set, the mixture was either (i) subjected to RNA extraction using the Qiagen QIAamp Viral RNA Mini Kit followed by subsequent testing by RT-qPCR (using the equivalent of 11.3 μl of swab diluent) or (ii) directly added to the RT-qPCR reaction, with or without a preheating step (5 minutes at 70°C, “NP sample + heat”). As a control, the indicated quantities of the CDC 2019-nCoV Positive Control SARS-CoV-2 synthetic RNA were spiked into M6 transport medium, purified using the QIAamp Viral RNA Mini Kit, and screened by RT-qPCR. NP swab samples from 7 additional donors were screened by direct RT-qPCR for SARS-CoV-2 RNA using the 2019-nCoV_N1 primer/probe set (B) or the 2019-nCoV_N2 primer/probe set (C), or for human RNase P RNA using the RNase P primer/probe set (D). NP swab samples from donors 1–4 were previously shown to contain SARS-CoV-2 RNA by standard clinical RT-qPCR, while donors 5–7 were negative. For each primer/probe set, 7 μl (A) or 3 μl (B–D) of NP swab diluent was tested in the RT-qPCR reaction per donor. For the N1 and N2 primer/probe sets, the fully synthetic SARS-CoV-2 RNA Control 2 from Twist Bioscience was loaded at serial 10-fold dilutions (A, 3 × 10 6 copies; B, 3 × 10 5 copies; C, 3 × 10 4 copies; D, 3 × 10 3 copies; E, 3 × 10 2 copies; F, 3 × 10 1 copies) as indicated in (B) and (C). NTC wells were included for each primer/probe set, and each was negative. For (B) and (C), the correlation coefficients ( R 2 ) of the standard curves were 0.999 and 0.995, respectively. The dashed line at cycle 40 in each graph indicates the limit of detection. CDC, Centers for Disease Control and Prevention; CT, cycle threshold; NP, nasopharyngeal; NTC, no template control; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: RNA Extraction, Quantitative RT-PCR, Positive Control, Purification, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection of SARS-CoV-2 RNA from NP swab diluent by direct RT-qPCR and the impact of heat and loading volume on assay sensitivity.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection sensitivity of direct RT-qPCR versus standard RT-qPCR on NP swabs containing a range of SARS-CoV-2 viral RNA loads.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 150 NP swab samples representing high (CT values less than 20), intermediate (CT values of 20–30), or low (CT values of more than 30) SARS-CoV-2 RNA loads as determined by standard clinical RT-qPCR at the University of Washington in Seattle (aqua circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was extracted from 30 μl of NP swab diluent that had been previously heated at 95°C for 10 minutes, and RNA representing 3 μl of the original diluent was used in RT-qPCR (purple circles) to allow a head-to-head comparison with direct RT-qPCR on the same quantity of NP swab diluent. The limit of detection (CT of 40) is denoted with a dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the mean of CT values detected in the clinical RT-qPCR assay with primer sets N1 and N2. Samples are ordered by the latter mean. The full dataset for this experiment and controls are provided in . CT, cycle threshold; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 60 NP swab samples representing low loads (CT of 27–36) of SARS-CoV-2 RNA as determined by standard clinical RT-qPCR at UW in Seattle (purple circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was newly extracted from 200 μl of NP swab diluent (aqua circles) and processed with the UW LDT to control for the effect of freeze/thaw cycles. The limit of detection (CT of 40) is denoted with a red dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the CT values detected in the 200-μl freshly extracted RT-qPCR assay with primer set N2. Samples are ordered by the CT value of freshly extracted 200-μl LDT samples. The full dataset for this experiment and controls are provided in . CT, cycle threshold; LDT, laboratory developed test; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; UW, University of Washington.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: RT-qPCR conditions.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: