Systems and methods for identifying viral contaminants
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsDavid Vanhoute, Steven Davis
Life of this patent
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Abstract
The present disclosure is directed to a streamlined sample preparation process, VERA (Viral Enrichment by Reducing Artifacts), to tilt total genomic material in favor of DNA/RNA viral genomes. This reduction of host genomic artifacts can be completed in <8 hours from sample acquisition. Using a rapid library preparation protocol (˜15 minutes) and real-time nanopore sequencing, potential viral contamination, for example RNA viral contamination, can be identified in less than one workday from sample acquisition.
Claims31
-18. (canceled)
An amplification-free method for identifying viral contaminant in a cell culture sample, comprising:
- (a) removing cellular debris from a lysed eukaryotic cells culture sample;
- (b) concentrating the cell culture sample via diafiltration to produce a retentate;
- (c) treating the retentate with more than one nuclease to degrade eukaryotic nucleic acids, wherein the more than one nuclease is one or more endonucleases and one or more RNases;
- (d) extracting viral nucleic acids from the nuclease-treated retentate;
- (e) preparing a library of the extracted viral nucleic acids for sequencing;
- and
- (f) sequencing the extracted viral nucleic acids from the prepared library without amplifying the extracted viral nucleic acids, thereby identifying the viral contaminant in the cell culture sample.
A system for amplification-free identification of a virus in a eukaryotic cell culture sample by reduction of host genomic material, comprising:
- (a) a mechanical device for lysing freeze-thawed eukaryotic cells from a cell culture sample, thereby lysing the cells;
- (b) a centrifuge or a filtration system for separating cellular debris from the lysed cells, thereby removing cellular debris from the cell culture sample;
- (c) a diafiltration system for concentrating the sample from step (b), thereby obtaining concentrated sample to produce a retentate;
- (d) at least one nuclease selected from the group consisting of an endonuclease and one or more RNase for treating the retentate with the nuclease, thereby degrading eukaryotic nucleic acids to reduce host genomic material;
- (e) a nucleic acid extraction kit for processing the nuclease-treated retentate, thereby extracting viral nucleic acids from the cell culture sample;
- (f) a library preparation kit for of the extracted viral nucleic acids, thereby obtaining a library of nucleic acids for sequencing;
- and
- (g) a sequencer for sequencing the extracted viral nucleic acids from the prepared library without amplifying the extracted viral nucleic acids, thereby identifying the virus in the cell culture sample.
Dependencies are inferred from the claim text.