Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Schematic illustrating SARS‐CoV‐2 genome and regions targeted by RT‐qPCR primers and probes. A. Schematic overview portrays the SARS‐CoV‐2 genome with RdRP and E gene regions magnified to show the locations of primers and probes of the original Charité protocol, vDetect (v1 and v2), and rTEST RT‐qPCR assays. F, forward primer; P, probe; R, reverse primer. The inset boxes (from left to right) illustrate a SARS‐CoV‐2 particle with labelled structural proteins and RNA, legend describing panel A and the primers and probes used in each test to detect RNase P subunit p30 (RPP30). B. Diagram compares the sequences of RdRP and E gene primers and probes for the original Charité protocol, vDetect (v.1) and vDetect v.2 and rTEST RT‐qPCR assays to the Wuhan reference sequence. The numbers written above the Wuhan reference sequence correspond to the start and end base positions of the sequence Reverse primer sequences are written in the reverse complement (rc). Magenta lines and letters represent mixed bases found in the primers and probes in the Charité protocol that were replaced with the correct bases in vDetect v1 (blue lines and letters). Red lines and letters signify LNA‐modified bases.

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: Quantitative RT-PCR, Sequencing, Modification

Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Optimization, analytical sensitivity and clinical performance of a rapid, RNA extraction‐free, multiplexed RT‐qPCR assay. A. Analytical sensitivity of the triplexed E, RdRP and RNase P assay in the rTEST COVID‐19 qPCR Allplex kit. B. Clinical performance of the rTEST COVID‐19 qPCR Allplex kit. C. Optimization of gargle sample input for a rapid, RNA extraction‐free, triplexed rTEST. D. Comparison of four different thermal profiles using 8 μl of gargle input volume in rapid, direct RT‐qPCR. E. Analytical sensitivity of the triplexed E, RdRP and RNase P assay in the RNA extraction‐free rTEST COVID‐19 qPCR Rapid kit. F. Clinical performance of the rTEST COVID‐19 qPCR Rapid kit. The dotted line at C t = 40 (panels A and E) serves as a threshold after which amplification is considered invalid. The dotted lines and shaded areas (panels B and F) indicate samples that were not detected by either the evaluation test, index test or both tests. C t , cycle threshold; E, envelope gene; ND, not detected within 45 cycles; NTC, no template control; RdRP, RNA‐dependent RNA polymerase.

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: RNA Extraction, Quantitative RT-PCR, Comparison, Amplification, Control

Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Schematic illustrating influenza A and B genome and regions targeted by RT‐qPCR primers and probes. (A) Schematic overview portrays the influenza A and B genome with PB1 and PA gene regions magnified to show the locations of primers and probes. Nucleotides labelled in red text indicate mixed bases in the consensus sequences for influenza A and B. BHQ2, black hole quencher 2; F, forward primer; HA, haemagglutinin; M, matrix protein; NA, neuraminidase; NP, nucleoprotein; NS, non‐structural protein; P, probe; PA, polymerase acidic protein; PB1, polymerase basic 1 protein; PB2, polymerase basic 2 protein; R, reverse primer; seg., segment; YY, Yakima Yellow ® .

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: Quantitative RT-PCR

Journal: Scientific reports

Article Title: NAxtra magnetic nanoparticles for low-cost, efficient isolation of mammalian DNA and RNA.

doi: 10.1038/s41598-023-46868-5

Figure Lengend Snippet: Figure 2. Nucleic acid (NA) extraction from 100 to 1,000,000 cells (HAP1), comparing the NAxtra-based method on KingFisher Flex (three independent NA extractions) to the AllPrep DNA/RNA/miRNA Universal Kit (two independent NA extractions). (A) NAs extracted from 1 = 1,000,000 and 2 = 500,000 cells, separated on a 0.4% agarose gel (left, 1% eluate applied for 1,000,000 cells, 2% eluate applied for 500,000 cells) with GeneRuler High Range DNA ladder (Thermo Scientific), or 1.2% agarose gel (right, 4% eluate applied for 1,000,000 cells, 8% eluate applied for 500,000 cells) with GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). Gel images have been inverted and cropped; original gels are presented in Supplementary Fig. 2. (B) Average NA yields (± 1 SD) from 500,000 and 1,000,000 cells, as measured by Quant-iT RNA/DNA assay (Invitrogen). (C) Average cycle threshold (Ct) values (± 1 SD) for DNA target (left; a 305 bp, single-locus genomic DNA amplicon from the KAPA Human Genomic DNA Quantification and QC Kit) and RNA target (right; ACTB) amplified by (RT-) qPCR of NA extracted from 100; 1000; 10,000 and 100,000 cells.

Article Snippet: Two different master mixes were used for each target: RealQ Plus 2 × Master Mix for Probe (Ampliqon) and Luna Universal Probe qPCR Master Mix (NEB), or One-Step Takyon Ultra Probe 4X MasterMix (Eurogentec) and 4X 1Step RT qPCR Probe Kit (highQu).

Techniques: Extraction, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR

Journal: Scientific reports

Article Title: NAxtra magnetic nanoparticles for low-cost, efficient isolation of mammalian DNA and RNA.

doi: 10.1038/s41598-023-46868-5

Figure Lengend Snippet: Figure 6. Nucleic acids (NA) extracted from adherent cells (HAP1) using the NAxtra-based method on KingFisher Flex (three independent NA extractions) compared to the AllPrep DNA/RNA/miRNA Universal Kit (two independent NA extractions). Average cycle threshold (Ct) values (± 1 SD) for a DNA target (top, MYC) and RNA target (bottom, ACTB) amplified by (RT-)qPCR of NAs extracted from 100 cells and 100,000 cells of HAP1. Two different master mixes were used for each target: RealQ Plus 2 × Master Mix for Probe (Ampliqon) and Luna Universal Probe qPCR Master Mix (NEB), or One-Step Takyon Ultra Probe 4X MasterMix (Eurogentec) and 4X 1Step RT qPCR Probe Kit (highQu).

Article Snippet: Two different master mixes were used for each target: RealQ Plus 2 × Master Mix for Probe (Ampliqon) and Luna Universal Probe qPCR Master Mix (NEB), or One-Step Takyon Ultra Probe 4X MasterMix (Eurogentec) and 4X 1Step RT qPCR Probe Kit (highQu).

Techniques: Amplification, Quantitative RT-PCR

Journal: Scientific reports

Article Title: NAxtra magnetic nanoparticles for low-cost, efficient isolation of mammalian DNA and RNA.

doi: 10.1038/s41598-023-46868-5

Figure Lengend Snippet: Figure 7. RNA isolated from triplicates of 1,000,000 cells (HAP1) using the NAxtra-based method compared to the MagMAX mirVana Total RNA isolation kit (Applied Biosystems) on KingFisher Duo Prime. Statistical analysis was performed by two-sided, unpaired t-tests, in which ns (non-significant) = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, and **** P ≤ 0.0001. (A) Total RNA separated on a 1.2% agarose gel (5% eluate applied) with GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). The gel image has been inverted and cropped; the original gel is presented in Supplementary Fig. 8. (B) Average total RNA yield (± 1 SD), as measured by Quant-iT RNA assay (Invitrogen) (C) Average cycle threshold (Ct) values (± 1 SD) for five miRNA targets; hsa- miR-24-3p, hsa-miR-122-5p, hsa-miR-210-5p, hsa-miR-455-5p and hsa-miR-1246, amplified by RT-qPCR. D) Average cycle threshold (Ct) values (± 1 SD) for an mRNA target (ACTB) amplified by RT-qPCR.

Article Snippet: Two different master mixes were used for each target: RealQ Plus 2 × Master Mix for Probe (Ampliqon) and Luna Universal Probe qPCR Master Mix (NEB), or One-Step Takyon Ultra Probe 4X MasterMix (Eurogentec) and 4X 1Step RT qPCR Probe Kit (highQu).

Techniques: Isolation, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR

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: AM, RK are founders of and employed by myPOLS Biotec GmbH, manufacturer of Volcano3G polymerase used in this manuscript.

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: AM, RK are founders of and employed by myPOLS Biotec GmbH, manufacturer of Volcano3G polymerase used in this manuscript.

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: AM, RK are founders of and employed by myPOLS Biotec GmbH, manufacturer of Volcano3G polymerase used in this manuscript.

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