Detection method using eukaryotic cells
Inventors
BARNELL, Erica • BARNELL, Andrew • KANG, Yiming • WURTZLER, Elizabeth
Assignees
Geneoscopy Inc Formerly Known Geneoscopy AS LLC • Geneoscopy Inc
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Publication Number
US-11479820-B2
Publication Date
2022-10-25
Expiration Date
Abstract
Provided herein are materials and methods for isolation of eukaryotic nucleic acid from a human or non-human animal stool sample. Also provided are methods of analysis of eukaryotic biomarkers present in a human or non-human animal stool sample.
Core Innovation
The invention relates to a method of isolating eukaryotic nucleic acid from a stool sample. The method forms a suspension by mixing the sample with a buffer, a surfactant and a ribonuclease inhibitor, and separates the suspension into a portion enriched for eukaryotic cells and a portion enriched for bacterial cells while retaining the eukaryotic cell enriched portion.
The retained portion enriched for eukaryotic cells is treated with a chaotropic agent and optionally a surfactant to form a lysate. The lysate is fractioned into a cell debris layer, a layer comprising eukaryotic nucleic acids and a lipid layer, and the layer comprising eukaryotic nucleic acids is collected, with the lipid layer optionally collected.
The described approach enables broad downstream biomarker detection and gene-expression analysis from stool-derived eukaryotic nucleic acids. It describes comparative expression level analysis of one or more biomarker genes versus controls to support risk assessment and clinical plan selection for colorectal cancer and gastrointestinal disorders, together with transcriptome analysis and signature development using PCR-related methods, NanoString-based approaches, microarray sequencing, molecular barcoding, probe capture, and sequencing workflows.
Claims Coverage
The independent claim provides a core workflow with five inventive features: preparing a suspension with buffer, surfactant, and ribonuclease inhibitor; separating into eukaryote-enriched versus bacteria-enriched portions; chaotropic lysis of the eukaryote-enriched portion; fractionating lysate into debris, nucleic-acid, and lipid layers; and collecting the eukaryotic-nucleic-acid-containing layer. No other independent claims are explicitly present in the provided content.
Stool suspension with buffer, surfactant, and ribonuclease inhibitor
Mixing the stool sample with a buffer, a surfactant and a ribonuclease inhibitor to form a suspension.
Eukaryotic-cell enriched versus bacterial-cell enriched separation
Separating the suspension into a portion enriched for eukaryotic cells and a portion enriched for bacterial cells and retaining the portion enriched for eukaryotic cells.
Chaotropic lysis of the eukaryotic-cell enriched portion
Adding a chaotropic agent and optionally a surfactant to the portion enriched for eukaryotic cells to form a lysate.
Fractionation into debris, eukaryotic nucleic acids layer, and lipid layer
Fractioning the lysate into a cell debris layer, a layer comprising eukaryotic nucleic acids and a lipid layer.
Collecting the eukaryotic nucleic acids layer
Collecting the layer comprising eukaryotic nucleic acids and optionally the lipid layer.
Across the provided independent claim, the coverage centers on enriching eukaryotic cells from stool, chaotropic lysing of the eukaryotic-enriched portion, fractionating the lysate into debris, nucleic-acid, and lipid layers, and collecting the layer comprising eukaryotic nucleic acids.
Stated Advantages
Improved RNA quality and eligibility for transcriptome analysis, as reported in examples.
Enables detection and gene-expression analysis using multiple downstream analysis options, including colorectal neoplasm biomarker panels and signature-based approaches.
Documented Applications
Risk assessment and clinical plan selection for colorectal cancer and gastrointestinal disorders by comparing expression levels or relative proportions of one or more biomarker genes to controls.
Application to stool samples from non-human cats and dogs, including gastrointestinal lymphoma and inflammatory bowel disease, for the described risk assessment and clinical plan selection.
Transcriptome analysis using stool-derived eukaryotic nucleic acids, with reported improvement in RNA quality and eligibility for transcriptome analysis.
Biomarker detection and gene-expression analysis for colorectal neoplasm biomarker panels using gene lists described in figures.
NanoString-based signature development in the context of the described biomarker and transcriptomic analysis.
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