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: