Journal: mSphere

Article Title: Targeted metatranscriptomic detection of viruses from floors for simultaneous evaluation of respiratory disease burden and viral variant identification

doi: 10.1128/msphere.00086-26

Figure Lengend Snippet: ( A ) Bubble chart showing the normalized read abundances of samples collected between the final week of November (week 4) 2022 and the final week of February (week 4) 2023 for all detected viruses in each sample. Samples are colored and ordered according to date, and the size of the bubble corresponds to abundance of normalized reads. HWA, hallway A; HWB, hallway B; NTC, no template control; OWB, outside waiting room B; PC, positive control; TRI, triage; WRA, waiting room A; WRB, waiting room B (a mixture of SARS-CoV-2, RSV, and influenza virus RNA). ( B ) Histogram showing the number of samples each virus was detected in (out of 38 swab samples). Samples were normalized using a scaling approach; the median number of viral target reads across all samples (not including controls) was determined, and a scaling factor for each sample was calculated compared to this median. The scaling factor was applied to all viral read counts within that sample.

Article Snippet: Influenza A, influenza B, and SARS-CoV-2 synthetic RNA controls were obtained from Twist Bioscience (catalog numbers 103001, 103003, and 103907, respectively).

Techniques: Control, Positive Control, Virus

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: (A) Schematic of the hybridization-based rolling circle amplification (hybRCA), performed entirely inside a microfluidic device by capturing the RNA target in streptavidin-coated agarose beads using biotinylated “capture” oligonucleotides – the biotinylated anchors. (B) Dilution series of SARS-CoV-2 control 2 RNA detected using HybRCA. The shaded region corresponds to the LoD of the method (average of the negative control plus three times the standard deviation; n = 3); representative images for 3 detected concentrations (300 copies per µL, 3000 copies per µL and 30 000 copies per µL, respectively); scale bars correspond to 100 µm for each image.

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Hybridization, Amplification, Control, Negative Control, Standard Deviation

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: (A) Schematic of the circle-to-circle amplification (C2CA) method used for RNA detection and variant profiling, using T4 RNA ligase 2 (T4Rnl2) as the RNA ligase of choice, combined with “chimeric” padlock probes (PLPs) in the first amplification round. Detection oligonucleotides (DO) with different fluorophores were used to detect the different SARS-CoV-2 variants: Cy3 for Wu (Wuhan strain); Cy5 for Alpha (variant B.1.1.7); and AF488 for beta (variant B.1.351). (B) Counts of amplification products (RCPs) labelled with each of the different used DOs when detecting various concentrations of each SARS-CoV-2 variant. Each value corresponds to the sum of four independent 300 × 300 µm 2 images of 10 µL of labelled solution in positively charged glass slides. (C) Average fluorescent intensity (au) measured in the microchannels of the µACE for the different used DOs when detecting the various SARS-CoV-2 variants. Each value corresponds to the average grey scale intensity of the bead-packed region, normalized for the fluorescence of the empty channel. The shaded region corresponds to the determined LoD.

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Amplification, RNA Detection, Variant Assay, Fluorescence

Journal: RSC Advances

Article Title: Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA

doi: 10.1039/d6ra00912c

Figure Lengend Snippet: Registered signal obtained for each different DO when detecting various samples containing various concentrations and SARS-CoV-2 variants using (A) positively charged glass slides or (B) µACE as a detection method. The solutions contain either (−) none of the variant, (+) 10 2 copies per µL of the variant, or (++) 10 4 copies per µL of the variant ( n = 3).

Article Snippet: Direct viral RNA detection on the microfluidic device using a single round of RCA was performed using synthetic control 2 SARS-CoV-2 RNA (GISAID ID: MN908947.3 ; Twist Bioscience), supplied at approximately 1 × 10 6 copies per μL.

Techniques: Variant Assay