Compositions and methods for detecting Yersinia pestis bacteria

• US 9,441,204 B2
• Filed: 04/03/2009
• Issued: 09/13/2016
• Est. Priority Date: 04/03/2008
• Status: Active Grant

• Current Assignee: Colorado School of Mines
• Original Assignee: Colorado School of Mines
• Inventors: Voorhees, Kent J., Luna, Leah G.
• Primary Examiner(s): KINSEY WHITE, NICOLE ERIN
• Application Number:
  US12/418,320
• Publication Number:
  US 20090258341A1
• Time in Patent Office: 2,720 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 2795/10211 Podoviridae
• CPC Class Codes: C12N 2795/10245 Special targeting system for viral vectors
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

Genetically modified bacteriophage and methods of using the same to detect bacterial types of interest are provided.

Claims

1. A method for determining the presence or absence of bacteria of a target bacterial type in a sample, comprising

(i) 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 and results in overexpression of the bacteriophage marker gene;

(ii) 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

(iii) assaying said bacteriophage exposed sample for the bacteriophage marker encoded by said bacteriophage marker gene to detect the protein expressed from the 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,

wherein said nucleic acid sequence encoding a bacteriophage marker encodes an endogenous head assembly protein, and wherein said expression control region affects overexpression of said bacteriophage marker gene encoding said endogenous head assembly protein,

wherein said bacteriophage marker encoded by said bacteriophage marker gene is not accessible for detection on intact progeny phage, and

wherein said target bacterial type is Yersinia pestis, and

wherein said endogenous head assembly protein is the 15.8 kDa φ

A1122 head assembly protein.

2. The method according to claim 1, further comprising the use of a negative control, wherein steps (i)-(iii) are repeated with a control sample known to lack bacteria of the target bacterial type to confirm that said assaying step does not detect the presence of said bacteriophage marker in the bacteriophage exposed sample in the absence of bacterial infection.

3. The method according to claim 1, wherein said assaying step involves lysis of the bacteria.

4. The method according to claim 1, wherein said bacteria of the target bacterial type are present in said sample at a concentration of less than about 1000 cells/ml.

5. The method according to claim 1, wherein said incubation step comprises a period of time less than the time required to initiate a second cycle of infection by progeny of said genetically modified bacteriophage.

6. The method according to claim 1, wherein said incubation step comprises a period of time less than the replication time of said genetically modified bacteriophage.

7. The method according to claim 1, wherein said incubation step comprises a period of time less than about 45 minutes.

8. The method according to claim 1, wherein the period of time from said contacting step to the result of said assaying step comprises a period of time less than about 45 minutes.

9. The method according to claim 1, wherein said assaying step involves the use of at least one antibody to detect said bacteriophage marker.

10. The method according to claim 9, wherein said antibody is a monoclonal antibody.

11. The method according to claim 9, wherein said antibody is present on a lateral flow immunochromatography (LFI) apparatus or a SILAS surface.

12. The method according to claim 1, wherein said target bacterial type is Yersinia pestis, said genetically modified bacteriophage is a genetically modified φ

A1122, said nucleic acid sequence encoding a bacteriophage marker encodes a φ

A1122 endogenous head assembly protein, said expression control region affects overexpression of said bacteriophage marker gene encoding said endogenous head assembly protein following infection of Yersinia pestis.

13. The method according to claim 12, wherein said recombinant bacteriophage marker gene comprises upstream gene φ

13-LaqI^q-pTrc-rbs-gene φ

13.