Journal: Frontiers in Public Health

Article Title: Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform

doi: 10.3389/fpubh.2022.1042647

Figure Lengend Snippet: Biomeme assay design, testing and validation workflow. Triplex assay development workflow. Using the Wuhan-Hu-1 isolate (accession no. MN908947.3) as the reference, whole genomes of SARS-CoV-2 variants were compared. The in silico framework consisted of identification of appropriate primers and probes and their subsequent suitability in triplex combinations. The primers were designed at regions of consensus sequences, and the probes were designed manually at target mutation sites. For the locked nucleic acid (LNA) probes, melting temperature predictions served as guides for mismatch discrimination. Candidate primers and probes were then aligned using BLAST to ensure specificity, and suitable triplex combinations were subsequently predicted. Identified triplexes were then tested and optimized initially using dsDNA templates (IDT gBlocks), followed by the use of in vitro -transcribed RNA templates. The optimized conditions were formulated into freeze-dried Go-Strips and Go-Plates for use on the Biomeme Franklin TM and standard 96-well RT-qPCR instruments.

Article Snippet: Initial testing and optimization were performed in singleplex reactions using gBlock templates, but then progressed to multiplex testing using synthetic SARS-CoV-2 gRNA commercially available from Twist Biosciences (San Francisco, CA, USA), as well as RNA extracted from patient specimens and stock cultures (once available).

Techniques: Biomarker Discovery, Assay Development, In Silico, Mutagenesis, In Vitro, Quantitative RT-PCR

Journal: Frontiers in Public Health

Article Title: Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform

doi: 10.3389/fpubh.2022.1042647

Figure Lengend Snippet: Serial dilution of extracted gRNA of different SARS-CoV-2 variants were used as templates in RT-qPCR reactions. The Cq values are plotted as a function of the concentrations. PCR efficiencies were derived from the slopes of the curves.

Article Snippet: Initial testing and optimization were performed in singleplex reactions using gBlock templates, but then progressed to multiplex testing using synthetic SARS-CoV-2 gRNA commercially available from Twist Biosciences (San Francisco, CA, USA), as well as RNA extracted from patient specimens and stock cultures (once available).

Techniques: Serial Dilution, Quantitative RT-PCR, Derivative Assay

Journal: Scientific Data

Article Title: Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States

doi: 10.1038/s41597-024-03969-8

Figure Lengend Snippet: Positive controls used in the study for SARS-CoV-2 targets and variants, Gene blocks are dsDNA molecules with the target sequence purchased from Integrated DNA Technologies (IDT, Coralville, Iowa).

Article Snippet: SARS-CoV-2 BA.4 + BA.5 + BQ* , Mutation in the S gene S:del69/70 , SARS-CoV-2 B.1.117 gRNA (ATCC VR-3326HK).

Techniques: Sequencing, Positive Control, Mutagenesis, Control, Blocking Assay

Journal: Scientific Data

Article Title: Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States

doi: 10.1038/s41597-024-03969-8

Figure Lengend Snippet: Forward and reverse primers, and probe sequences for detection of SARS-CoV-2 nucleic acids in this study.

Article Snippet: SARS-CoV-2 BA.4 + BA.5 + BQ* , Mutation in the S gene S:del69/70 , SARS-CoV-2 B.1.117 gRNA (ATCC VR-3326HK).

Techniques: Sequencing