Methods and systems for rapid detection of microorganisms using infectious agents
AssigneeLaboratory Corporation of America Holdings
InventorsDwight Lyman Anderson, Jose S. Gil, Ben Barrett Hopkins, Stephen Erickson
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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.
Claims30
A recombinant bacteriophage, comprising a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene;
- and
- (ii) a promoter controlling transcription of the indicator gene;
- wherein the promoter is a bacteriophage late promoter,
- and wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein, encodes a free soluble protein and does not yield a fusion protein.
A method for detecting a microorganism of interest in a sample comprising the steps of:
- incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage comprises a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising a luciferase gene and a bacteriophage late promoter controlling transcription of the luciferase gene, wherein the luciferase gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and wherein expression of the luciferase gene during bacteriophage replication following infection of host bacteria results in a free soluble luciferase protein product;
- and,
- detecting the luciferase protein product, wherein positive detection of the luciferase protein product indicates that the microorganism of interest is present in the sample.
A system for rapid detection of a microorganism of interest in a sample, comprising:
- a component for incubating the sample with a recombinant bacteriophage comprising a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene;
- and
- (ii) a promoter controlling transcription of the indicator gene;
- wherein the promoter is a bacteriophage late promoter,
- and wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein, encodes a free soluble protein and does not yield a fusion protein;
- a component for capturing the microorganism of interest on a solid support, wherein the microorganism of interest is bound to the solid support such that a washing step will not remove the microorganism of interest from the solid support;
- and
- a component for detecting expression of the indicator gene.
A kit for rapid detection of a microorganism of interest in a sample, comprising:
- a component for incubating the sample with a recombinant bacteriophage comprising a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising
- (i) an indicator gene;
- and
- (ii) a promoter controlling transcription of the indicator gene;
- wherein the promoter is a bacteriophage late promoter,
- and wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein, encodes a free soluble protein and does not yield a fusion protein;
- a component for capturing the microorganism of interest on a solid support, wherein the microorganism of interest is bound to the solid support, such that a washing step will not remove the microorganism of interest from the solid support;
- and a component for detecting the free soluble protein expressed from the indicator gene.
A method for preparing a recombinant bacteriophage, comprising inserting into a late gene region of a bacteriophage genome a genetic construct comprising an indicator gene and a bacteriophage late promoter controlling transcription of the indicator gene, wherein the indicator gene comprises a luciferase gene, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein, wherein the indicator gene encodes a free soluble protein, and wherein the indicator gene does not yield a fusion protein.
A recombinant bacteriophage comprising a genetic construct inserted into a late gene region of the bacteriophage genome, the genetic construct comprising:
- (i) an indicator gene encoding a free soluble indicator protein product;
- and
- (ii) a promoter controlling transcription of the indicator gene;
- wherein the promoter is a bacteriophage late promoter,
- wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein,
- and wherein the recombinant bacteriophage is purified to be free of the indicator protein product.
Dependencies are inferred from the claim text.