Compositions and Methods for Detecting Bacteria
First Claim
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1. A method for determining the presence or absence of bacteria of a target bacterial type in a sample, comprising(i) providing a sample;
- (ii) contacting the sample with genetically modified bacteriophage that are selective for the target bacterial type under conditions that allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample, thereby producing a bacteriophage exposed sample, wherein said genetically modified bacteriophage comprise a recombinant bacteriophage marker gene comprising a nucleic acid sequence encoding a bacteriophage marker operably linked to an expression control region that affects expression of said bacteriophage marker gene during the late eclipse or early latent period following infection of said bacteria of the target bacterial type by said genetically modified bacteriophage;
(iii) incubating said bacteriophage exposed sample for a period of time sufficient to allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample; and
(iv) assaying said bacteriophage exposed sample for the bacteriophage marker encoded by said bacteriophage marker gene, wherein said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection, and wherein the presence of said bacteriophage marker indicates the presence of said bacteria of the target bacterial type in said sample.
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Abstract
Genetically modified bacteriophage and methods of using the same to detect bacterial types of interest are provided.
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23 Claims
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1. A method for determining the presence or absence of bacteria of a target bacterial type in a sample, comprising
(i) providing a sample; -
(ii) contacting the sample with genetically modified bacteriophage that are selective for the target bacterial type under conditions that allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample, thereby producing a bacteriophage exposed sample, wherein said genetically modified bacteriophage comprise a recombinant bacteriophage marker gene comprising a nucleic acid sequence encoding a bacteriophage marker operably linked to an expression control region that affects expression of said bacteriophage marker gene during the late eclipse or early latent period following infection of said bacteria of the target bacterial type by said genetically modified bacteriophage; (iii) incubating said bacteriophage exposed sample for a period of time sufficient to allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that may be present in said sample; and (iv) assaying said bacteriophage exposed sample for the bacteriophage marker encoded by said bacteriophage marker gene, wherein said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection, and wherein the presence of said bacteriophage marker indicates the presence of said bacteria of the target bacterial type in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22)
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21. A genetically modified bacteriophage selective for a target bacterial type, comprising a recombinant bacteriophage marker gene, which marker gene comprises a nucleic acid sequence encoding a bacteriophage marker operably linked to an expression control region that is capable of affecting expression of said bacteriophage marker during the late eclipse or early latent period following infection of bacteria of said target bacterial type.
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23. A method for determining the resistance or susceptibility of a target bacterial type to an antibiotic, comprising:
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(i) dividing a primary sample containing bacteria of said target bacterial type into a first and a second sample; (ii) adding antibiotic to said first sample alone; (iii) contacting said first and second samples with genetically modified bacteriophage that are selective for the target bacterial type under conditions that allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type that are present in said second sample and may be present in said first sample, thereby producing first and second bacteriophage exposed samples, wherein said genetically modified bacteriophage comprise a recombinant bacteriophage marker gene comprising a nucleic acid encoding a bacteriophage marker operably linked to an expression control region that effects expression of said bacteriophage marker gene during the late eclipse or early latent period following infection of said bacteria of the target bacterial type by said genetically modified bacteriophage; (iv) incubating said bacteriophage exposed samples for a period of time sufficient to allow said genetically modified bacteriophage to infect said bacteria of the target bacterial type present in said second sample; (v) assaying said bacteriophage exposed samples for the bacteriophage marker encoded by said bacteriophage marker gene, wherein said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection, wherein the presence of said bacteriophage marker indicates the presence of said bacteria of the target bacterial type in said samples; and (vi) comparing the results of said assaying step for said first and second bacteriophage exposed samples, wherein decreased bacteriophage marker in said first bacteriophage exposed sample compared to said second bacteriophage exposed sample indicates that said target bacterial type is sensitive to said antibiotic.
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Specification