Devices and methods for detecting microorganisms using recombinant reproduction-deficient indicator bacteriophage
AssigneeLaboratory Corporation of America Holdings
InventorsStephen Erickson, Jose S. Gil, Minh Mindy Bao Nguyen, Wendy S. Hahn
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Abstract
Disclosed herein are compositions, methods, kits and systems for rapid detection of microorganisms using a reproduction-deficient indicator bacteriophage. The specificity of such reproduction-deficient indicator bacteriophage for binding and infecting particular microorganisms of interest allows targeted and sensitive detection of a microorganism of interest.
Claims26
A reproduction-deficient recombinant phage, comprising an indicator gene inserted into a late gene region of the phage genome, wherein one or more late genes required for virion assembly are deleted or altered in the recombinant phage, thereby rendering the recombinant phage reproduction-deficient, and wherein the recombinant phage is capable of specifically infecting a microorganism of interest.
A composition comprising at least two reproduction-deficient recombinant phages, each comprising an indicator gene inserted into a late gene region of the phage genome, wherein one or more late genes required for virion assembly are deleted or altered in the recombinant phage, thereby rendering the recombinant phages reproduction-deficient, and wherein the recombinant phages are capable of specifically infecting one or more microorganism of interest.
A method of preparing a recombinant phage, comprising:
- selecting a parent phage that specifically infects a target microorganism;
- altering or deleting one or more late genes of the phage required for virion assembly to generate a recombinant reproduction-deficient phage;
- transforming an engineered strain of the target microorganism capable of expressing a product of the gene mutated in the reproduction-deficient phage with a homologous recombination (HR) plasmid comprising an indicator gene and HR sequences flanking the indicator gene and homologous to a desired sequence in the parent phage;
- infecting the transformed target microorganism with the parent phage or the reproduction-deficient parent phage, allowing HR to occur between the HR plasmid and the genome or the parent phage or the recombinant reproduction-deficient phage;
- and
- isolating a particular clone of recombinant phage that is both reproduction-deficient and is capable of expressing a product of the indicator gene.
Dependencies are inferred from the claim text.