Methods for producing mutant bacteriophages for the detection of listeria
AssigneeLaboratory Corporation of America Holdings
InventorsJose S. Gil, Dwight L Anderson, Stephen Erickson, Minh Mindy Bao Nguyen
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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.
Claims29
A method of producing a mutant phage with expanded host range, comprising:
- (i) preparing a plurality of 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 plurality of the co-culture mixtures;
- (iii) incubating the plurality of the co-culture mixtures and the phage strain under bacterial culture conditions;
- (iv) collecting a phage lysate from each of the plurality of the co-culture mixtures;
- (v) pooling the collected phage lysates from each of the plurality of the co-culture mixtures;
- (vi) assaying the pooled phage lysates to determine if host phage of the phage strain has expanded;
- and,
- (vii) isolating the mutant phage with expanded host range,
- wherein the mutant bacteriophage with expanded host range is a recombinant mutant bacteriophage with expanded host range comprising an indicator gene inserted into a late gene region of genome.
A method of producing a mutant phage with expanded host range, comprising:
- (i) preparing a plurality of 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 plurality of the co-culture mixtures;
- (iii) incubating the plurality of the co-culture mixtures and the phage strain under bacterial culture conditions;
- (iv) collecting a phage lysate from each of the plurality of the co-culture mixtures;
- (v) pooling the collected phage lysates from each of the plurality of the co-culture mixtures:
- (vi) assaying the pooled phage lysates to determine if host phage of the phage strain has expanded;
- and,
- (vii) isolating the mutant phage with expanded host range,
- wherein the mutant phage with expanded host range is capable of infecting multiple serotypes of Listeria monocytogenes.
A method of producing a mutant phage with expanded host range, comprising:
- (i) preparing a plurality of 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 plurality of the co-culture mixtures;
- (iii) incubating the plurality of the co-culture mixtures and the phage strain under bacterial culture conditions;
- (iv) collecting a phage lysate from each of the plurality of the co-culture mixtures;
- (v) pooling the collected phage lysates from each of the plurality of the co-culture mixtures;
- (vi) assaying the pooled phage lysates to determine if host phage of the phage strain has expanded;
- and,
- (vii) isolating the mutant phage with expanded host range,
- wherein the phage strain in step (ii) is a bacteriophage of Caudovirales order.
Dependencies are inferred from the claim text.