# US 2020/0399614 A1 · Methods for Producing Mutant Bacteriophages for the Detection of Listeria > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399614 A1 - Document ID: US20200399614A1 - Status: Published application - Kind: A1 - Application number: 16/905,607 - Filed: 2020-06-18 - Published: 2020-12-24 - Earliest claimed: 2019-06-21 - Assignees: Laboratory Corporation of America Holdings - Applicants: Laboratory Corporation of America Holdings - Inventors: Jose S. Gil; Dwight L Anderson; Stephen Erickson; Minh Mindy Bao Nguyen - Web page: https://patentgrep.com/patent/US20200399614A1 ## CPC - A61P 31/04: Antibacterial agents - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C07K 14/005: from viruses - C12N 1/205: Bacterial isolates - C12N 2795/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2795/00051: Methods of production or purification of viral material - C12N 2795/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2795/10043: viral genome or elements thereof as genetic vector - C12N 2795/10051: Methods of production or purification of viral material - C12N 2795/10121: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2795/10143: viral genome or elements thereof as genetic vector - C12N 2795/10151: Methods of production or purification of viral material - C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales} - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract Disclosed herein are methods for the production of mutant bacteriophages with altered host range. Additionally, disclosed herein are methods and systems for rapid detection of microorganisms such as Listeria spp. 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 Listeria-specific bacteriophage, allows detection of a specific microorganism, such as Listeria spp. and an indicator signal may be amplified to optimize assay sensitivity. ## Claims ### Claim 1 A method of producing a mutant bacteriophage with expanded host-range comprising: (i) preparing a series of first co-culture mixtures of varying ratios comprising a host bacterial strain and a target-host bacterial strain; (ii) adding a phage strain to each of the first co-culture mixtures; (iii) incubating the first co-culture mixtures and the phage strain under bacterial culture conditions; (iv) collecting a phage lysate from each of the plurality of first-co-cultures; (v) pooling the phage lysates from each of the plurality of first co-cultures; (vi) assaying phage lysates to determine if the bacteriophage host-range has expanded; and (vii) isolating a mutant phage with expanded host-range. ### Claim 2 (depends on 1) The method of claim 1 wherein the selected phage strain infects the host bacterial strain and not the target-host bacterial strain. ### Claim 3 (depends on 1) The method of claim 1 wherein the selected phage strain is selected from A511, P100, LMA4, and LMA8. ### Claim 4 (depends on 1) The method of claim 1 wherein the host bacterial strain is Listeria monocytogenes 19115. ### Claim 5 (depends on 1) The method of claim 1 wherein the target-host bacterial strain is Listeria monocytogenes 51782. ### Claim 6 (depends on 1) The method of claim 1, wherein the series of the plurality of first co-cultures comprises a ratio of 1:0, 9:1, 1:1, 1:9, and 0:1 of the host bacterial strain:the target-host bacterial strain. ### Claim 7 (depends on 1) The method of claim 1, wherein steps are repeated until the assayed phage shows evidence of an expanded host-range. ### Claim 8 (depends on 1) The method of claim 1, further comprising passing the resulting phage cultivated in each co-culture ratio. ### Claim 9 A recombinant mutant bacteriophage with expanded host range comprising an indicator gene inserted into a late gene region of the bacteriophage genome, wherein the recombinant bacteriophage has been mutated to infect a host bacterium and a target-host bacterium. ### Claim 10 (depends on 9) The recombinant mutant bacteriophage of claim 9, wherein the recombinant bacteriophage is derived from wild-type A511, P100, LMA4 or LMA8 bacteriophage. ### Claim 11 (depends on 9) The recombinant mutant bacteriophage of claim 9, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates signal upon reaction with a substrate. ### Claim 12 (depends on 9) The recombinant mutant bacteriophage of claim 9, further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region includes a bacteriophage late gene promoter and a ribosomal entry site. ### Claim 13 (depends on 9) A cocktail composition comprising at least two different types of recombinant mutant bacteriophages, wherein at least one of the recombinant bacteriophages comprises an indicator gene according to claim 9.