Journal: Frontiers in Microbiology

Article Title: An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

doi: 10.3389/fmicb.2021.749783

Figure Lengend Snippet: Workflow of ultrafast one-step qRT-PCR with comparison with conventional qRT-PCR for SARS-CoV-2 detection.

Article Snippet: Using synthetic SARS-CoV-2 RNA from ATCC as the model, we found that 0.4 U of SpeedSTAR HS DNA Polymerase (in 10 μL of qRT-PCR reaction mixture) in the one-step qRT-qPCR assay can detect down to 25 copies of N1 and N2 of SARS-CoV-2 RNA ( ).

Techniques: Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

doi: 10.3389/fmicb.2021.749783

Figure Lengend Snippet: Limit of detection of ultrafast one-step qRT-PCR assay. SARS-Cov-2 synthetic RNA genome was used as a model sample. RNA templates were series diluted in the range of 25–1 × 10 5 copies. Ultrafast one-step qRT-PCR assay detects both N1 (A) and N2 (B) regions of the nucleocapsid gene of SARS-CoV-2.

Article Snippet: Using synthetic SARS-CoV-2 RNA from ATCC as the model, we found that 0.4 U of SpeedSTAR HS DNA Polymerase (in 10 μL of qRT-PCR reaction mixture) in the one-step qRT-qPCR assay can detect down to 25 copies of N1 and N2 of SARS-CoV-2 RNA ( ).

Techniques: Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

doi: 10.3389/fmicb.2021.749783

Figure Lengend Snippet: Cycle threshold (Ct values) of N1 and N2 correlation of ultrafast one-step qRT-PCR with FDA-approved assay “Xpert ® Xpress SARS-CoV-2” for SARS-CoV-2 positive samples. (A) Ct value of N1 and (B) Ct value of N2 from the 30 SARS-CoV-2 positive samples.

Article Snippet: Using synthetic SARS-CoV-2 RNA from ATCC as the model, we found that 0.4 U of SpeedSTAR HS DNA Polymerase (in 10 μL of qRT-PCR reaction mixture) in the one-step qRT-qPCR assay can detect down to 25 copies of N1 and N2 of SARS-CoV-2 RNA ( ).

Techniques: Quantitative RT-PCR

Journal: medRxiv

Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

doi: 10.1101/2020.07.16.20150250

Figure Lengend Snippet: A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E , ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2X saliva stabilization solution, heated at 95°C for 10 minutes, and then spiked with in vitro transcribed SARS-CoV-2 RNA at the indicated concentrations. 4 μL of this was added to a master mix containing primers and NEB’s WarmStart LAMP 2x Master Mix in a final reaction volume of 20 μL. Reactions were incubated at 65°C and a fluorescence reading was taken every 30 seconds. EvaGreen was used to monitor amplification products in real-time (X-axis) using a QuantStudio3 quantitative PCR machine. There are 9 lines for each of the three primer sets because three concentrations of spiked in SARS-CoV-2 RNA were each tested in triplicate (0, 400, 800 copies / μL saliva). The saliva samples without SARS-CoV-2 RNA spike in are shown as flat lines. When concentrations are given herein, denominator refers to the raw, pre-diluted saliva sample. The normalized change in fluorescence signal (ΔRn) is shown on the Y-axis. B) Saliva mixed 1:1 with 2X saliva stabilization solution was heated (95°C for 10 minutes) and then spiked with SARS-CoV-2 RNA at the indicated concentrations. Replicates were tested by RT-LAMP with the control RNaseP primer set and three distinct SARS-CoV-2 primer sets (AS1E, ORF1e, and CU-N2). All samples scored positive except those boxed, which are saliva samples that contain no SARS-CoV-2 RNA, as expected.

Article Snippet: Heat-inactivated SARS-CoV-2 virion control (ATCC #VR-1986HK) was obtained and its concentration of 3.75×10 5 virions/μL was confirmed using quantitative RT-PCR in conjunction with both the synthetic SARS-CoV-2 RNA control and a DNA plasmid control containing a region of the N gene from the SARS-CoV-2 genome.

Techniques: In Vitro, Incubation, Fluorescence, Amplification, Real-time Polymerase Chain Reaction

Journal: medRxiv

Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

doi: 10.1101/2020.07.16.20150250

Figure Lengend Snippet: A) Saliva samples were spiked with the indicated concentrations of heat-inactivated SARS-CoV-2 virions (top) before being diluted 1:1 with 2X saliva stabilization solution. Samples were then heated at 95°C for 10 minutes and subjected to RT-LAMP at 65°C for 30 minutes in 6 replicates. Each panel represents a unique primer set (listed at the bottom of each panel). The table at the bottom shows a summary of positive reactions observed (yellow). Red box indicates the determined RT-LAMP limit of detection (LOD). B) Saliva samples were spiked with heat-inactivated SARS-CoV-2 virions at a concentration of 200 virions/μL (the limit of detection of our assay) before being diluted 1:1 with 2X saliva stabilization solution. Samples were then heated at 95°C for 10 minutes and 20 replicates of RT-LAMP with the indicated primer sets were incubated at 65°C for 30 minutes. The table at the bottom shows a summary of positive reactions (yellow). Red box indicates our selection of primer sets to advance to subsequent analysis.

Article Snippet: Heat-inactivated SARS-CoV-2 virion control (ATCC #VR-1986HK) was obtained and its concentration of 3.75×10 5 virions/μL was confirmed using quantitative RT-PCR in conjunction with both the synthetic SARS-CoV-2 RNA control and a DNA plasmid control containing a region of the N gene from the SARS-CoV-2 genome.

Techniques: Concentration Assay, Incubation, Selection

Journal: medRxiv

Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

doi: 10.1101/2020.07.16.20150250

Figure Lengend Snippet: A) We re-analyzed university saliva samples that had been previously analyzed for SARS-CoV-2 using quantitative RT-PCR with a primer set against the N gene of SARS-CoV-2 (see ). The remaining saliva was mixed 1:1 with 2X saliva stabilization solution (without Proteinase K) and re-tested using RT-LAMP. The results of RT-LAMP are compared to relative saliva viral load determined by quantitative RT-PCR. The figure shows the distribution of the viral load of all 278 positive saliva samples separated by the corresponding RT-LAMP reaction results with either the AS1E or CU-N2 primer-set. B) Saliva TwoStep RT-LAMP test sensitivity as a function of the cycle threshold (Ct) from the quantitative RT-PCR results of the corresponding SARS-CoV-2 positive saliva samples. C) A summary of the sensitivity and specificity of the Saliva TwoStep test from the blinded sample evaluation described above and shown in (top), and from both the data in panel A (bottom).

Article Snippet: Heat-inactivated SARS-CoV-2 virion control (ATCC #VR-1986HK) was obtained and its concentration of 3.75×10 5 virions/μL was confirmed using quantitative RT-PCR in conjunction with both the synthetic SARS-CoV-2 RNA control and a DNA plasmid control containing a region of the N gene from the SARS-CoV-2 genome.

Techniques: Quantitative RT-PCR

Journal: medRxiv

Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

doi: 10.1101/2020.07.16.20150250

Figure Lengend Snippet: (A) Matched nasal swabs and saliva from 54 individuals were analyzed (all of whom were SARS-CoV-2 positive at the time that these samples were collected, as verified by saliva direct quantitative RT-PCR test). Nasal swab samples from the same individuals were collected within 2 days of positive saliva test, and tested using the EUA Quidel Direct Lyra RT-PCR test. The saliva samples from those same individuals were tested with the Saliva TwoStep test. Data points represent individuals (n = 54), and the corresponding test result is color-coded: positive, yellow; negative, grey. (B) Positive test agreement between Saliva TwoStep and the two comparator tests. The nature of the sample used by each test (nasal swab or saliva), and the test chemistry (quantitative RT-PCR or RT-LAMP) are delineated.

Article Snippet: Heat-inactivated SARS-CoV-2 virion control (ATCC #VR-1986HK) was obtained and its concentration of 3.75×10 5 virions/μL was confirmed using quantitative RT-PCR in conjunction with both the synthetic SARS-CoV-2 RNA control and a DNA plasmid control containing a region of the N gene from the SARS-CoV-2 genome.

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: medRxiv

Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

doi: 10.1101/2020.07.16.20150250

Figure Lengend Snippet: Person provides 1 mL of saliva, and 1 mL of 2X saliva stabilization solution is then added to it. (This sample can be processed immediately or stored in the refrigerator at 4°C for at least 4 days.) The mixture is heated at 95°C for 10 minutes. This step serves to neutralize the pH of saliva, liberate viral RNA from virions in the saliva, and inactivate virions for safe handling (although appropriate safety precautions should always be taken). We have determined that performing a heating step at 95°C for 30 minutes in a water bath before addition of the saliva stabilization solution also works equally well. However, in this case Proteinase K must be left out of that solution. Lower half) Step 2: Detect Virus . 2 μL of stabilized saliva from step 1 is pipetted into each of three test tubes pre-filled with the RT-LAMP master mix and primers. The only thing different between the three tubes is the primer set included, with each set targeting either the human positive control RNA or a region of SARS-CoV-2 RNA, as indicated. After incubation, the reaction will turn from pink to yellow if the target RNA is present in saliva. An example of a positive and a negative test are shown. Graphic by Annika Rollock.

Article Snippet: Heat-inactivated SARS-CoV-2 virion control (ATCC #VR-1986HK) was obtained and its concentration of 3.75×10 5 virions/μL was confirmed using quantitative RT-PCR in conjunction with both the synthetic SARS-CoV-2 RNA control and a DNA plasmid control containing a region of the N gene from the SARS-CoV-2 genome.

Techniques: Positive Control, Incubation