Method for detecting concentrations of a target bacterium that uses phages to infect target bacterial cells

• US 7,972,773 B2
• Filed: 01/23/2007
• Issued: 07/05/2011
• Est. Priority Date: 04/12/2002
• Status: Expired due to Fees

• Current Assignee: Colorado School of Mines
• Original Assignee: Colorado School of Mines
• Inventors: Rees, Jon C., Madonna, Angelo J., Voorhees, Kent J.
• Primary Examiner(s): Lucas; Zachariah
• Assistant Examiner(s): White; Nicole Kinsey
• Application Number:
  US11/626,326
• Publication Number:
  US 20070275370A1
• Time in Patent Office: 1,624 Days
• Field of Search: None
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

The invention is directed to a method for detecting low concentrations of bacteria in liquid solution that may or may not be complex liquid solutions. In one embodiment, immunomagnetic separation (IMS) is used to separate target bacterium that may be in a liquid mixture from other constituents in the mixture. A low concentration of a bacteriophage for the target bacteria is subsequently used to infect target bacterial cells that have been captured using the IMS technique. If at least a certain concentration of target bacterium are present, the bacteriophage will multiply to a point that is detectable. Matrix assisted laser desorption ionization/time-of-flight-mass spectrometry (MALDI/TOF-MS) is then used to produce a mass spectrum that is analyzed to determine if one or more proteins associated with the bacteriophage are present, thereby indirectly indicating that target bacterium were present in the liquid mixture.

Claims

1. A method for determining if a target bacterium is present in a liquid solution when the target bacterium is below the detection limit for a particular detection technology comprising:

combining a first quantity of a bacteriophage with a liquid suspension comprising a first quantity of target bacteria to infect at least some of said target bacteria and multiply the number of bacteriophage in said liquid solution, wherein each of said first quantity of target bacteria and the first quantity of bacteriophage is at a concentration below the detection limit for said detection technology; and

analyzing at least a portion of said liquid suspension by said detection technology to determine if a biomarker for said bacteriophage is present in order to determine the presence of said target bacterium in said liquid solution.

2. A method, as claimed in claim 1, wherein:

said step of analyzing comprises performing an immuno analysis.

3. A method, as claimed in claim 1 further comprising separating said target bacterium from said liquid suspension before said combining step.

4. A method, as claimed in claim 3, wherein:

said step of separating comprises using immuno-magnet beads coated with antibodies for said target bacterium.