Methods and systems for the rapid detection of cronobacter using infectious agents
Inventors
Nguyen, Minh Mindy Bao • Gil, Jose S. • Erickson, Stephen • Anderson, Dwight Lyman • Stach, Jessica
Assignees
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Publication Number
US-12146182-B2
Publication Date
2024-11-19
Expiration Date
Abstract
Disclosed herein are methods and systems for rapid detection of microorganisms such as Cronobacter 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 Cronobacter-specific bacteriophage, allows detection of a specific microorganism, such as Cronobacter spp. and an indicator signal may be amplified to optimize assay sensitivity.
Core Innovation
The invention relates to a method for detecting Cronobacter spp. in a sample, specifically including a sample of infant formula, by incubating the sample with a cocktail composition comprising at least two different types of recombinant bacteriophages. Each recombinant bacteriophage is derived from a Cronobacter-specific bacteriophage and includes an indicator gene inserted into a late gene region of the bacteriophage genome, with a phage late gene promoter exogenous to the Cronobacter-specific bacteriophage upstream of the indicator gene.
The indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, and expression of the indicator gene results in an indicator protein product. Detection is performed by detecting the indicator protein product produced by the recombinant bacteriophage, where positive detection indicates that Cronobacter spp. is present in the sample.
The at least two different recombinant bacteriophages comprise at least one first recombinant bacteriophage derived from a podovirus Cronobacter-specific bacteriophage and at least one second recombinant bacteriophage derived from a myovirus Cronobacter-specific bacteriophage. The disclosure further characterizes embodiments in which the indicator protein product is luciferase and detection includes measuring bioluminescent emissions from a luciferase-catalyzed reaction.
Claims Coverage
The claims cover a primary detection method for Cronobacter spp. in an infant formula sample using a cocktail of at least two recombinant Cronobacter-specific bacteriophages, with the indicator gene expression driven by an exogenous phage late gene promoter upstream of a late gene region. Four inventive features are identified.
Cocktail of at least two recombinant Cronobacter-specific bacteriophages with exogenous phage late promoter
Incubating the sample with a cocktail composition comprising at least two different types of recombinant bacteriophages, wherein each recombinant bacteriophage is derived from a Cronobacter-specific bacteriophage comprising an indicator gene inserted into a late gene region of bacteriophage genome and, upstream of the indicator gene, a phage late gene promoter exogenous to the Cronobacter-specific bacteriophage.
Non-fusion indicator gene expression from late gene region
The indicator gene is not contiguous with a gene encoding a structural bacteriophage protein and does not yield a fusion protein, wherein expression of the indicator gene results in an indicator protein product.
Indicator protein detection indicating presence of Cronobacter spp.
Detecting the indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Cronobacter spp. is present in the sample, wherein the sample is a sample of infant formula.
Podovirus- and myovirus-derived recombinant phage combination
The at least two different types of recombinant bacteriophages comprise at least one first recombinant bacteriophage derived from a podovirus Cronobacter-specific bacteriophage, and at least one second recombinant bacteriophage derived from a myovirus Cronobacter-specific bacteriophage.
Overall claim coverage centers on a phage-based detection method that uses a cocktail of recombinant Cronobacter-specific bacteriophages, each carrying an indicator gene inserted into a late gene region under an exogenous phage late gene promoter, where a non-fusion indicator protein is produced and detected to indicate presence of Cronobacter spp. in an infant formula sample, using both podovirus-derived and myovirus-derived recombinant phages.
Stated Advantages
Provides rapid detection for Cronobacter spp. in infant formula samples based on detection of an indicator protein product produced by recombinant bacteriophages.
Uses luciferase-based bioluminescent emissions as a detection approach when the indicator protein product is luciferase.
Enables sensitive detection at very low bacterium counts, including detection as few as one bacterium and up to 10 bacteria in a standard-sized food safety sample.
Supports constrained total time to results through an enrichment period window described as less than 24 hours.
Includes performance characterized by signal-to-background thresholds and robustness across test conditions and matrices, including matrix studies versus ISO 22964:2006 and FDA BAM Ch. 29 showing no significant differences overall.
Documented Applications
Detecting Cronobacter spp. in a sample of infant formula using a cocktail of recombinant Cronobacter-specific bacteriophages and detecting an indicator protein product to indicate presence.
Detection performed in matrix studies using powdered infant formula and compared against ISO 22964:2006 and FDA BAM Ch. 29 reference methods.
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