Methods and systems for rapid detection of microorganisms using infectious agents
Inventors
Anderson, Dwight Lyman • Gil, Jose S. • Hopkins, Ben Barrett • Erickson, Stephen
Assignees
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Publication Number
US-12006531-B2
Publication Date
2024-06-11
Expiration Date
Abstract
Disclosed herein are methods and systems for rapid detection of microorganisms in a sample, without culturing for enrichment of the microorganism. A modified bacteriophage is also disclosed which comprises a non-native indicator gene in the late gene region. The indicator product is not a fusion protein. The specificity of infectious agents allows a specific microorganism to be targeted, and an indicator signal may be amplified to optimize assay sensitivity.
Core Innovation
The invention relates to detecting a bacterium of interest in a sample by incubating at least one bacterium with a plurality of a bacteriophage that is genetically engineered to have an indicator gene inserted into a late gene region of the bacteriophage. The indicator gene encodes a luciferase protein that is expressed during replication of the bacteriophage, and the luciferase protein does not form a fusion protein with a native gene product.
The detection step detects the luciferase protein, and detection of the luciferase protein indicates that the bacterium is present in the sample. In the described embodiments, luciferase expression occurs during phage replication and/or lysis as the recombinant bacteriophage replicates, producing a non-fusion luciferase indicator product that can be detected.
The disclosure supports rapid microorganism detection without traditional enrichment culturing, including assay formats such as filter-based approaches and capture-first approaches that use antibodies and binding components. The incubating step is performed at a temperature that is at least 37 degrees Celsius and no greater than 45 degrees Celsius.
Claims Coverage
The independent claim covers detecting a bacterium of interest by using genetically engineered bacteriophages carrying a non-fusion luciferase indicator gene inserted in a late gene region/class III region, expressing luciferase during phage replication, incubating within a defined 37–45 °C range, and detecting luciferase to indicate presence of the bacterium. The dependent claims refine the luciferase expression control, omit bacterial enrichment culturing, add sample isolation/capture prior to phage contact, and narrow to specific engineered promoter/host/phage examples, including T4, and a binding-agent isolation mechanism.
Genetically engineered bacteriophage with late-region luciferase indicator gene
Incubating at least one bacterium with a plurality of a bacteriophage genetically engineered to have an indicator gene inserted into a late gene region of the bacteriophage, wherein the indicator gene encodes a luciferase protein such that the luciferase protein is expressed during the replication of the bacteriophage.
Non-fusion luciferase indicator expression
Expressing luciferase during replication of the bacteriophage such that the luciferase protein does not form a fusion protein with a native gene product.
Temperature-controlled incubation for engineered phage infection
Performing the incubating step at a temperature that is at least 37 degrees Celsius and no greater than 45 degrees Celsius.
Luciferase detection indicates bacterium presence
Detecting the luciferase protein, wherein detection of the luciferase protein indicates that the bacterium is present in the sample.
Viral capsid promoter control and luciferase exclusion from fusion incorporation
Controlling luciferase protein expression by a viral capsid promoter and constraining the luciferase such that it is not incorporated into the bacteriophage structure.
Omission of culturing bacterial enrichment
Performing the method without culturing the sample for bacterial enrichment.
Isolation of bacterium from other sample components before contacting phage
Isolating a bacterium from other components in a sample before contacting it with a plurality of bacteriophages.
Binding-agent isolation using a solid support
Isolating a bacterium by binding the bacterium to a binding agent, including using a binding agent bound to a solid support.
Use of T4 bacteriophage
Using T4 bacteriophage.
Across the independent claim and refinements, the core coverage centers on engineered bacteriophages carrying an indicator gene (luciferase) inserted into a late gene region so luciferase is expressed during phage replication without forming a luciferase fusion product. Presence of the target bacterium is indicated by detection of luciferase following incubation in the specified 37–45 °C range, with dependent claims further refining expression control, excluding enrichment culturing, adding sample isolation/capture using binding agents and solid support, and specifying T4 phage.
Stated Advantages
Rapid microorganism detection without traditional enrichment culturing.
Enables detection of very low microorganism levels, including single cells, within short total assay times.
Allows multiple assay formats, including filter-based approaches and capture-first approaches using antibodies and binding components.
Provides specificity based on capture of a targeted bacterium of interest.
Allows detection using luciferase signal as an indicator of bacterium presence.
Documented Applications
Detecting E. coli O157:H7 using antibody capture in the disclosed hybrid immuno-phage (HIP) style approach.
Filter-based and 96-well/“No Concentration” assay formats for microorganism detection in samples, including vegetable wash matrix.
Using capture-first approaches involving antibodies, beads, or filters prior to phage indicator detection.
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