Methods and systems for rapid detection of microorganisms using infectious agents
AssigneeLaboratory Corporation of America Holdings
InventorsJose S. Gil, Stephen Erickson, Ben Barrett Hopkins, Minh Mindy Bao Nguyen, Dwight Lyman Anderson
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Abstract
Disclosed herein are methods and systems for rapid detection of microorganisms in a sample. A genetically modified bacteriophage is also disclosed which comprises an indicator gene in the late gene region. The specificity of the bacteriophage, such as CBA120, allows detection of a specific microorganism, such as E. coli O157:H7, and an indicator signal may be amplified to optimize assay sensitivity.
Claims18
A recombinant bacteriophage comprising an indicator gene inserted into a late gene region of the bacteriophage CBA120 genome, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and wherein expression of the indicator gene results in an indicator protein product.
A method of preparing a recombinant indicator bacteriophage comprising:
- selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, wherein the wild-type bacteriophage is CBA120 and the target pathogenic bacterium is E. coli O157:H7;
- preparing a homologous recombination plasmid/vector comprising an indicator gene, wherein preparing a homologous recombination plasmid/vector comprises:
- determining the natural nucleotide sequence in the late region of the genome of the selected bacteriophage;
- annotating the genome and identifying the major capsid protein gene of the selected bacteriophage;
- designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene;
- and
- incorporating the sequence designed for homologous recombination into a plasmid/vector;
- transforming the homologous recombination plasmid/vector into target pathogenic bacteria;
- infecting the transformed target pathogenic bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid/vector and the bacteriophage genome;
- and
- isolating a particular clone of recombinant bacteriophage.
A method for detecting E. coli O157:H7 in a sample comprising:
- incubating the sample with a recombinant CBA120 bacteriophage, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage genome, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and wherein expression of the indicator gene results in an indicator protein product;
- and
- detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that E. coli O157:H7 is present in the sample.
A kit for detecting E. coli O157:H7 comprising a recombinant bacteriophage derived from CBA120, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage genome, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and wherein expression of the indicator gene results in an indicator protein product.
A system for detecting E. coli O157:H7 comprising a recombinant bacteriophage derived from CBA120, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage genome, wherein the indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and wherein expression of the indicator gene results in an indicator protein product.
Dependencies are inferred from the claim text.