Journal: medRxiv

Article Title: Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification

doi: 10.1101/2020.05.19.20103150

Figure Lengend Snippet: Schematic overview of the SARS-CoV-2 genome. The target sequences for the N1 primers and probe are marked in red and green, respectively. The R2 primer binding sequence is underlined. Sequences divergence between SARS-CoV-2 and SARS-CoV-1 genomes are highlighted in blue. B) Performance of Volcano3G polymerase was compared to Taq polymerase using plasmid DNA or in vitro transcribed RNA as template (5000 viral genome equivalents). C) Determination of the linear dynamic range for the Volcano3G protocol with or without an additional primer (R2) for optimized reverse transcription at a final concentration of 250 nM. In vitro transcribed RNA containing the Sars-CoV-2 N amplicon was serially diluted in the range from 1×10 6 copies to 10 copies. D) Limit of detection (LOD) was assessed with serial dilutions ranging from 20 to 1 copy per reaction (n = 6 for each dilution). The fraction of positive reactions (y-axis) were plotted against the log-transformed number of RNA copies per reaction. Addition of R2 primer enhances the performance at lower copy-numbers. E) Amplification curves showing the performance of Volcano3G on isolated RNA from two COVID-19 patients in presence or absence of R2.

Article Snippet: Throughout this study high-temperature RT-PCR on RNA purified from either the in vitro transcribed viral genome fragment or the swab material was performed with the Volcano3G RT-PCR Probe 2x Master Mix (in short: Volcano3G) (myPOLS Biotec, Konstanz, Germany) using the CDC-approved N1 primer/probe mix from Integrated DNA Technologies (IDT, San Diego, CA, USA).

Techniques: Binding Assay, Sequencing, Plasmid Preparation, In Vitro, Concentration Assay, Amplification, Transformation Assay, Isolation

Journal: medRxiv

Article Title: Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification

doi: 10.1101/2020.05.19.20103150

Figure Lengend Snippet: A ) RNA was isolated from nasopharyngeal- and throat swab samples (n = 43) and SARS-CoV-2 and RNAseP were detected using the Volcano3G protocol. N1 amplicon (blue), RNaseP gene (gray). Water was used as a non-template control (light gray). B) Identical samples were processed in parallel in an accredited diagnostic lab using the Allplex 2019-nCoV assay from Seegene. Direct comparison of assay results reveals 100% concordance of Volcano3G with the reference assay. C) Cq values obtained with Volcano3G were lower than those obtained with the reference assay (ΔCq = 6.4 +/− 0.78). D) For each positive patient sample, the Cq values obtained with both assays were plotted against each other for linear regression analysis. A highly significant correlation of Volcano3G with the reference assay was observed (r 2 = 0.98, p<0.0001).

Article Snippet: Throughout this study high-temperature RT-PCR on RNA purified from either the in vitro transcribed viral genome fragment or the swab material was performed with the Volcano3G RT-PCR Probe 2x Master Mix (in short: Volcano3G) (myPOLS Biotec, Konstanz, Germany) using the CDC-approved N1 primer/probe mix from Integrated DNA Technologies (IDT, San Diego, CA, USA).

Techniques: Isolation, Amplification, Diagnostic Assay

Journal: medRxiv

Article Title: Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification

doi: 10.1101/2020.05.19.20103150

Figure Lengend Snippet: A) Nasopharyngeal- and throat swab samples (prepared in water) were added directly as template for RT-qPCR using the Volcano3G protocol. Representative amplification curves of patients with high (dark blue), medium (medium blue) and low Cq as well as negative patients (light blue) are shown. B) RNA was isolated from the remaining patient material and analysed in an accredited diagnostic lab using the Allplex 2019-nCoV assay from Seegene. C) The Cq values of each patient sample are compared between the reference protocol and the Volcano3G direct approach. Dotted red line indicates the cut-off, were the assay loses sensitivity. D) For each positive patient sample, the Cq values obtained with both assays were plotted against each other for linear regression analysis (r 2 = 0.779, p<0.0001) E) RTPCR analysis of 100 copies of in vitro transcribed RNA spiked with varying amounts of pooled patient material from 5 confirmed negative patients. F) RT-PCR analysis of confirmed COVID-19 patient samples with high Cq values were analysed in a larger volume. G) Four confirmed COVID-19 patient samples and one negative control were used directly as in A). The reference cq-values obtained by standard RT-PCR from purified RNA (upper row) and the cq-values obtained by high temperature RT-PCR with Volcano3G polymerase (loer row) are given. After completion of PCR cycling, the reaction tubes were photographed on a blue light transilluminator (470 nm). Positive samples emitted a distinct green fluorescence visible by the naked eye.

Article Snippet: Throughout this study high-temperature RT-PCR on RNA purified from either the in vitro transcribed viral genome fragment or the swab material was performed with the Volcano3G RT-PCR Probe 2x Master Mix (in short: Volcano3G) (myPOLS Biotec, Konstanz, Germany) using the CDC-approved N1 primer/probe mix from Integrated DNA Technologies (IDT, San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Amplification, Isolation, Diagnostic Assay, Reverse Transcription Polymerase Chain Reaction, In Vitro, Negative Control, Purification, Fluorescence