Methods and materials for detecting contaminated food products
Inventors
Smith, Kenneth D. • Yazvenko, Nina • Smit, Mariya
Assignees
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Publication Number
US-10167521-B2
Publication Date
2019-01-01
Expiration Date
Abstract
This document provides methods and materials for detecting contaminated food products. For example, methods and materials for using an enzymatic amplification cascade of restriction endonucleases to detect nucleic acid of a microorganism or virus (e.g., a pathogen) within a sample (e.g., food product sample) being tested, thereby assessing a food product for possible contamination are provided.
Core Innovation
The invention relates to a kit for assessing a food product for contamination by detecting target nucleic acid present within a microorganism or virus. A probe nucleic acid includes a nucleotide sequence complementary to a sequence of a target nucleic acid, and hybridization forms a double-stranded portion comprising a restriction endonuclease cut site, with the probe nucleic acid comprising a restriction endonuclease.
The system further includes signal expansion nucleic acid comprising an amplifying restriction endonuclease, a label, and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the restriction endonuclease of the probe nucleic acid. This arrangement provides a restriction-endonuclease-based signal expansion process tied to the probe/target hybridization event and an enzymatic amplification cascade of restriction endonucleases.
Detection modalities include fluorescence, colorimetric, and electrochemical readouts, and the described approach includes continuous near real-time fluorescent detection of live/dead microorganisms via oxygen-consumption culturing in combination with restriction endonuclease-based target nucleic-acid detection. The document states applications to specific microorganisms and viruses by targeting respective nucleic-acid sequences.
Claims Coverage
The document identifies one independent kit claim, covering a contamination-assessing kit with two core inventive features: a target-complementary probe nucleic acid engineered to create a restriction endonuclease cut site upon hybridization, and a signal expansion nucleic acid carrying an amplifying restriction endonuclease and a label associated with a corresponding restriction endonuclease cut-site double-stranded portion.
Hybridization-generated restriction endonuclease cut site in a probe nucleic acid
A probe nucleic acid comprising a nucleotide sequence complementary to a sequence of a target nucleic acid present within a microorganism or virus, wherein at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site, and wherein said probe nucleic acid comprises a restriction endonuclease.
Signal expansion nucleic acid with amplifying restriction endonuclease and labeled cut-site double-stranded portion
Signal expansion nucleic acid comprising an amplifying restriction endonuclease, a label, and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said restriction endonuclease of said probe nucleic acid.
The inventive coverage centers on a two-part nucleic-acid system: hybridization creates a double-stranded nucleic-acid portion containing a restriction endonuclease cut site in a probe nucleic acid, and a corresponding signal expansion nucleic acid includes an amplifying restriction endonuclease and a label carried with a matching cut-site double-stranded portion to expand and detect contamination.
Stated Advantages
Amplification improves detection limits by orders of magnitude.
Continuous near real-time fluorescent detection is described in combination with live/dead assessment via oxygen consumption culturing.
Provides presence/absence detection and potentially quantitative readouts based on labeled products generated by an amplifying restriction endonuclease cascade.
Documented Applications
Assessing a food product for contamination.
Detecting target microorganisms and viruses including E. coli O157:H7, Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Shigella dysenteriae, Vibrio parahaemolyticus, E. coli, and Norwalk virus.
Live/dead microbial assessment via oxygen-consumption culturing combined with restriction endonuclease-based target nucleic-acid detection using fluorescent detection.
Live-vs-dead discrimination via enrichment/culture and RNA targeting.
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