Methods and systems for rapid detection of Listeria using infectious agents - Patents.com

Abstract

Disclosed herein are methods and systems for the rapid detection of microorganisms (e.g., Listeria spp.) in a sample. Genetically modified bacteriophages containing indicator genes in the late gene region are also disclosed. The specificity of bacteriophages, such as Listeria-specific bacteriophages, allows for the detection of specific microorganisms, such as Listeria spp., and the indicator signal can be amplified to optimize assay sensitivity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 798,248, filed January 29, 2019. The disclosures of U.S. Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, and 15 / 409,258 are incorporated by reference herein in their entireties.

[0002] FIELD OF THEINVENTION The present invention relates to compositions, methods, systems and kits for the detection of microorganisms using infectious agents. [Background technology]

[0003] background There is strong importance in improving the speed and sensitivity for the detection of bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause substantial morbidity in humans and domestic animals, as well as huge economic losses. Also, microbial detection is a high priority for the U.S. Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), as well as the U.S. Department of Agriculture (USDA), in light of the outbreaks of life-threatening or fatal diseases caused by the ingestion of food contaminated with certain microorganisms (e.g., Listeria spp., Salmonella spp., or Staphylococcus spp.).

[0004] In particular, Listeria spp. are known to cause a potentially serious infectious disease, listeriosis. Listeria spp. (e.g., L. monocytogenes) are typically transmitted through ingestion of contaminated food products. L. monocytogenes is a gram-positive bacterium commonly associated with the contamination of food products, including, but not limited to, milk, seafood, poultry, and meat. Foodborne illnesses (e.g., listeriosis) can be prevented by detecting contaminated foods before they are available to consumers.

[0005] Traditional microbiological tests for detecting bacteria rely on non-selective and selective enrichment cultures followed by plating on selective media and further testing to confirm suspected colonies. Such procedures can take as long as 7 days. For example, traditional tests for the detection of Listeria spp. in food products are complex and time-consuming, requiring a 24-48 hour enrichment period followed by a more tedious test with a total detection time ranging from 5-7 days. Various rapid methods have been investigated and introduced into practice to reduce the time requirements. However, these methods have drawbacks. For example, polymerase chain reaction (PCR) tests also include an amplification step and are therefore capable of both very high sensitivity and selectivity; economically they are limited to small sample sizes. With dilute bacterial suspensions, most small subsamples are cell-free and therefore purification and / or tedious enrichment steps are still required.

[0006] The time required for traditional biological enrichment is dictated by the growth rate of the target bacterial population of the sample, by the effect of the sample matrix, and by the required sensitivity. Due to the time required for cultivation, these methods can take up to 8 days depending on the organism to be identified and the source of the sample. This lag time is generally inadequate since contaminated food, water (or other products) may have already made its way into livestock or humans. Furthermore, the rise of antibiotic-resistant bacteria and biodefense concerns has made the rapid identification of bacterial pathogens in water, food and clinical samples a vital priority worldwide.

[0007] Thus, there is a need for faster, simpler and more sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms. Summary of the Invention [Means for solving the problem]

[0008] Abstract Embodiments of the present invention include compositions, methods, systems and kits for the detection of microorganisms, such as Listeria spp. The present invention can be embodied in a variety of ways.

[0009] In some aspects, the invention includes a recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Listeria-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified bacteriophage genome derived from a bacteriophage that specifically recognizes Listeria spp. In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage specifically infects one or more Listeria spp. In one embodiment, the recombinant bacteriophage is capable of distinguishing Listeria spp. in the presence of other types of bacteria. In some embodiments, the recombinant bacteriophage specifically recognizes Listeria monocytogenes.

[0010] In some embodiments of the recombinant indicator bacteriophage, the indicator gene may be codon-optimized and may encode a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. Some recombinant bacteriophages further comprise an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene may be a naturally occurring gene, such as an Oplophorus luciferase gene, a firefly luciferase gene, a Lucia luciferase gene, or a Renilla luciferase gene, or the luciferase gene may be an engineered gene, such as NANOLUC®.

[0011] Also disclosed herein is a method for preparing a recombinant indicator bacteriophage. Some embodiments include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into a target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid / vector and the genome of the bacteriophage, and isolating a specific clone of the recombinant bacteriophage. In some embodiments, the selected wild-type bacteriophage is a Listeria-specific bacteriophage.

[0012] In some embodiments, the step of preparing a homologous recombination plasmid / vector includes determining the native nucleotide sequence in the late region of the genome of the selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence comprising a codon-optimized indicator gene, and incorporating the sequence designed for homologous recombination into the plasmid / vector. The step of designing the sequence may include inserting a gene construct comprising a phage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene. In some embodiments, the phage late gene promoter is an exogenous promoter that is different from any endogenous promoter in the phage genome. Thus, in some methods, the homologous recombination plasmid comprises a bacteriophage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene.

[0013] Some embodiments of the present invention are compositions comprising the recombinant indicator bacteriophages described herein. For example, the compositions may comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions, methods, systems and kits of the present invention may comprise a cocktail of at least one recombinant bacteriophage for use in detecting microorganisms such as Listeria spp.

[0014] In some embodiments, the invention encompasses a method for detecting a microorganism of interest in a sample, comprising incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage contains an indicator gene inserted into a late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the microorganism of interest is present in the sample.

[0015] In some embodiments of the method for preparing a recombinant indicator bacteriophage, the wild-type bacteriophage is a Listeria spp.-specific bacteriophage and the target pathogenic bacterium is Listeria spp. In some embodiments, isolating a specific clone of the recombinant bacteriophage comprises a limiting dilution assay to isolate clones that exhibit expression of the indicator gene.

[0016] Other aspects of the invention include methods for detecting bacteria (e.g., Listeria spp.) in a sample comprising incubating the sample with a recombinant bacteriophage derived from a Listeria-specific bacteriophage and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Listeria spp. is present in the sample. In some embodiments, the invention includes methods for detecting Listeria spp. using recombinant bacteriophage derived from a bacteriophage that targets Listeria spp. The sample may be a food sample or a water sample. In some embodiments, samples include environmental samples (e.g., sponges and swabs of surfaces or equipment for bacterial monitoring in factories and other processing facilities).

[0017] In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions favoring growth for an enrichment period of 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, or 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In some embodiments, the sample is not enriched prior to detection. In some embodiments, the total time to result is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. In some embodiments, the signal to background ratio generated by detecting the indicator is at least 2.0 or at least 2.5 or at least 3.0. In some embodiments, the method detects as few as 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample of standard size for the food safety industry.

[0018] Further embodiments include systems and kits for detecting Listeria spp., said systems or kits comprising recombinant bacteriophages derived from Listeria-specific bacteriophages. Some embodiments further comprise a substrate for reacting with an indicator to detect a soluble protein product expressed by said recombinant bacteriophage. These systems or kits may include features described with respect to the bacteriophages, compositions and methods of the invention. In yet other embodiments, the invention includes a non-transitory computer readable medium for use with a method or system according to the invention. [Brief description of the drawings]

[0019] The invention can be better understood with reference to the non-limiting drawings.

[0020] [Figure 1] 1 shows an indicator phage construct according to one embodiment of the present invention illustrating the insertion of a genetic construct comprising a luciferase gene, a bacteriophage late gene promoter, and a ribosome binding site (RBS) inserted into the late (class III) region of a bacteriophage. The promoter shown is upstream of an endogenous late gene (e.g., the gene for major capsid protein (MCP)) in addition to or separate from the endogenous late gene promoter.

[0021] [Diagram 2]Figure 2 shows the genome of bacteriophage LMA4, a myovirus (related to Listeria phage LMTA-94) obtained from sewage. A hypothetical gene homologous to the putative prohead protease p85 protein is upstream of the major capsid gene in the late gene region, which consists of cps, a structural gene encoding a virion protein. Following the cps is a transcription terminator, followed by a homolog to the LMTA-94 tail sheath protein (tsh). Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, so long as the late gene promoter and / or other similar control elements are used.

[0022] [Diagram 3]Figure 3 shows two homologous recombination plasmid construct designs with the luciferase gene used to construct recombinant phages, along with approximately several hundred base pairs of matching phage sequence upstream and downstream of the insertion site to facilitate homologous recombination. NANOLUC® luciferase is inserted into the pCE104 Gram-positive shuttle vector plasmid backbone with a dedicated phage late gene promoter and an upstream untranslated region containing a ribosome entry site. pCE104.HR.A511.NanoLuc.v2 was used to construct recombinants for Pecentumviruses A511, LMA4 and LMA8. pCE104.HR.LP-ES1.NanoLuc was used to construct Homburvirus LP-ES1. Each construct consisted of a fragment of the major capsid protein gene (cps) followed by 500 bp of homologous sequence consisting of the late gene promoter. This added the luciferase gene, as well as approximately 258 bp of downstream matching sequence for homologous recombination for pCE104.HR.A511.NanoLuc.v2 and 500 bp downstream for pCE104.HR.LP-ES1.NanoLuc, in addition to the endogenous late gene promoter upstream of the major capsid protein in the phage genome. All recombinants used the P100 viral late gene promoter instead of the T4 late gene promoter.

[0023] [Figure 4] FIG. 4 describes a filter plate assay for detecting bacteria of interest using modified bacteriophages according to one embodiment of the present invention, in which bacteria and recombinant phages are incubated on the filter plate and the indicator protein is directly detected after production of progeny bacteriophage without removal of the incubation medium.

[0024] [Diagram 5]5A and 5B show data from an embodiment of a Listeria detection assay using recombinant bacteriophage specific for Listeria to detect Listeria in spiked sponges using 10 mL spiked medium (5A) or 90 mL spiked medium (5B).

[0025] [Figure 6] FIG. 6 shows data from an embodiment of a Listeria detection assay that uses recombinant bacteriophage specific for Listeria to detect Listeria in environmental surface swab sponge samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention Disclosed herein are compositions, methods and systems that exhibit surprising sensitivity for the detection of microorganisms of interest (e.g., Listeria spp.) in test samples (e.g., biological, food, water and environmental samples). Detection can be achieved in shorter time frames than previously thought possible using genetically modified infectious agents in assays performed with minimal enrichment times (during which the microorganisms can potentially grow). Also surprising is the success of using potentially high multiplicities of infection (MOI), or high concentrations of plaque forming units (PFU), to incubate with the test sample. Such high phage concentrations (PFU / mL) were previously claimed to be detrimental in bacterial detection assays because they were claimed to cause "lysis from without". However, high concentrations of phage may facilitate discovery, binding and infection of small numbers of target cells.

[0027] The compositions, methods, systems, and kits of the invention may include infectious agents for use in the detection of microorganisms such as Listeria spp. In certain embodiments, the invention may include compositions comprising recombinant bacteriophage having an indicator gene inserted into the late gene region of the bacteriophage. In certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene (i.e., class III) region of the bacteriophage. The bacteriophage may be derived from a Listeria spp.-specific bacteriophage, or another wild-type or engineered bacteriophage. In some embodiments, the recombinant bacteriophage is constructed from at least one of LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A bacteriophage.

[0028] In some embodiments, the compositions, methods, systems and kits of the present invention may comprise a cocktail of at least one recombinant bacteriophage for use in the detection of microorganisms such as Listeria spp.

[0029] In some aspects, the present invention includes a method for detecting a microorganism of interest. The method may use an infectious agent for the detection of the microorganism of interest (e.g., Listeria spp.). For example, in certain embodiments, the microorganism of interest is Listeria spp. and the infectious agent is a bacteriophage that specifically infects Listeria spp. Thus, in certain embodiments, the method may include detection of a bacterium of interest in a sample by incubating the sample with a recombinant bacteriophage that infects the bacterium of interest. In certain embodiments, the recombinant bacteriophage includes an indicator gene. The indicator gene may, in certain embodiments, be inserted into the late gene region of the bacteriophage such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in the production of an indicator protein product. The method may include detecting the indicator protein product, where positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample. In some embodiments, the indicator protein is soluble.

[0030] In certain embodiments, the invention may include a system. The system may include at least some of the compositions of the invention. The system may also include at least some of the components for carrying out the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the invention may include a system for rapid detection of a microorganism of interest, such as Listeria spp., in a sample, the system including a component for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; and a component for detecting the indicator moiety. In yet other embodiments, the invention includes software for use with the method or system.

[0031] Thus, some embodiments of the present invention address the need by using a bacteriophage-based method to amplify a detectable signal indicative of the presence of bacteria. In certain embodiments, as few as 10 bacteria are detected. The principles applied herein can be applied to the detection of a variety of microorganisms. Because of the many binding sites for infectious agents on the surface of microorganisms, the ability to generate progeny during infection, and the potential for high-level expression of the encoded indicator moiety, the infectious agent or indicator moiety may be more easily detectable than the microorganism itself. In this way, embodiments of the present invention can achieve very large signal amplification from even a single infected cell.

[0032] Aspects of the invention take advantage of the high specificity of binding agents capable of binding to particular microorganisms, such as binding components of infectious agents, as a means to detect and / or quantitate specific microorganisms in a sample. In some embodiments, the invention takes advantage of the high specificity of infectious agents (e.g., bacteriophages).

[0033] In some embodiments, detection is accomplished through an indicator moiety associated with a binding agent specific for the microorganism of interest. For example, an infectious agent may include an indicator moiety, such as a gene encoding a soluble indicator. In some embodiments, the indicator may be encoded by the infectious agent, such as a bacteriophage, which is referred to as an indicator phage.

[0034] Some embodiments of the invention disclosed and described herein take advantage of the discovery that a single microorganism can bind a specific recognition factor (e.g., a phage). After infection and replication of the phage, the progeny phage can be detected via an indicator moiety expressed during phage replication. This principle allows for the amplification of an indicator signal from one or a few cells based on the specific recognition of a microorganism surface receptor. For example, by exposing as few as 10 bacterial cells to multiple phages and then allowing high-level expression of the encoded indicator gene product during the phage amplification and replication, the indicator signal is amplified so that the bacteria can be detected.

[0035] Embodiments of the methods and systems of the present invention may be applied to the detection and quantification of various microorganisms (e.g., bacteria) in various environments, including, but not limited to, detection of pathogens from food samples, water samples, and commercial samples. The methods of the present invention provide rapid, high detection sensitivity and specificity. In some embodiments, detection is possible within a single replication cycle of the bacteriophage, which is unexpected.

[0036] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques, are well known and commonly used in the art. Known methods and techniques are generally performed by conventional methods well known in the art and as described in various general and more specific references discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with laboratory procedures and techniques described herein are well known and commonly used in the art.

[0037] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0038] As used herein, the terms "a," "an," and "the" can refer to one or more, unless otherwise noted.

[0039] Use of the term "or" is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0040] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among samples.

[0041] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface onto which a biomolecule can be attached. For example, a solid support can be an assay well (i.e., for example, a microtiter plate or a multiwell plate), or a solid support can be a filter, an array, or a location on a mobile support (e.g., beads) or membrane (e.g., a filter plate, latex particles, paramagnetic particles, or lateral flow strips).

[0042] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction can be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently bound) to a solid support.

[0043] As used herein, "analyte" refers to a molecule, compound, or cell being measured. An analyte of interest may, in certain embodiments, interact with a binding agent. As described herein, the term "analyte" may refer to a protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like.

[0044] The term "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicator moiety" refers to a molecule that can be measured in a quantitative assay. For example, an indicator moiety can include an enzyme that can be used to convert a substrate into a product that can be measured. An indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that results in the emission of bioluminescence. Alternatively, an indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0045] As used herein, "bacteriophage" or "phage" includes one or more of the viruses of bacteria. In this disclosure, the terms "bacteriophage" and "phage" include viruses such as mycobacteriophage (e.g., for TB and paraTB), mycophage (e.g., for fungi), mycoplasma phage, and any other term that refers to a virus that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and use the organism to replicate itself. Here, "microscopic" means one millimeter or less in maximum dimension. Bacteriophages are viruses that have evolved in nature to use bacteria as a means to replicate themselves. Phages do this by attaching themselves to bacteria, injecting their DNA (or RNA) into the bacteria, and inducing the bacteria to replicate the phage hundreds or even thousands of times. This is also called phage amplification.

[0046] As used herein, "late gene region" refers to a region of a viral genome that is transcribed late in the viral life cycle. The late gene region typically contains the most abundantly expressed genes, such as structural proteins that assemble into the bacteriophage particle. Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, class I (e.g., RNA polymerase) is transcribed early, at 4-8 minutes, and class II, at 6-15 minutes, from 8 minutes post-infection until lysis, with the timing of II and III overlapping. A late promoter is a promoter that is naturally located and active in such a late gene region.

[0047] As used herein, "enrichment culture" refers to traditional culture (e.g., incubation in a medium favorable for microbial growth) and should not be confused with other possible uses of the term "enrichment" (e.g., enrichment by removing a liquid component of a sample to concentrate the microorganisms contained therein) or other forms of enrichment that do not involve traditional promotion of microbial growth. Enrichment cultures over a period of time may be used in some embodiments of the methods described herein.

[0048] As used herein, "recombinant" means a gene that is not found anywhere else. "Modified" refers to genetic (i.e., nucleic acid) modification, usually performed in a laboratory, to combine genetic material that is not native to the host. This term is used interchangeably herein with the term "modified."

[0049] As used herein, "RLU" refers to the relative units of light emitted, as measured by a luminometer (e.g., GLOMAX® 96) or similar instrument that detects light. For example, detection of the reaction between luciferase and an appropriate substrate (e.g., NANOLUC® and NANO-GLO®) is often reported in RLUs detected.

[0050] As used herein, "time to results" refers to the total amount of time from the start of sample incubation to when a result is generated. Time to results does not include any confirmatory testing time. Data collection can occur any time after the result is generated.

[0051] sample Each of the method and system embodiments of the present invention can allow for rapid detection and quantification of microorganisms in a sample. For example, methods according to the present invention can be performed in a shortened period of time with superior results.

[0052] Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food or water borne pathogens.

[0053] The sample may be liquid, solid, or semi-solid. The sample may be a swab of a solid surface. Samples may include filters from environmental materials (e.g., water samples), or air or aerosol samples from cyclone collectors. The sample may be a sample of vegetables, meat, fish, poultry, peanut butter, processed foods, infant formula, milk powder, tea, starch, eggs, milk, cheese, or other dairy products.

[0054] In some embodiments, samples may be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples, including but not limited to, milk and juice, may be assayed directly. Samples may be diluted or suspended in solutions, including but not limited to, buffered solutions or bacterial culture media. Samples that are solid or semi-solid may be suspended in liquid by chopping, mixing, or macerating the solid in the liquid. Samples should be maintained within a pH range that promotes bacteriophage attachment to the host bacterial cells. Samples may also be assayed for divalent and monovalent cations (Na +, Mg 2+ , and Ca 2+ The sample should contain an appropriate concentration of various antigens, including but not limited to, IgG ...

[0055] In some embodiments of the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. For example, during the step in which the bacteriophage is attaching to the bacterial cells, it is preferred to maintain the sample at a temperature that promotes bacteriophage attachment. During the step in which the bacteriophage is replicating in the infected bacterial cells or lysing such infected cells, it is preferred to maintain the sample at a temperature that promotes bacteriophage replication and lysis of the host. Such a temperature is at least about 25 degrees Celsius (C), more preferably about 35°C or less, and most preferably about 30°C.

[0056] The assay may include a variety of suitable control samples, for example, a control sample containing no bacteriophage or a control sample containing bacteriophage without bacteria may be assayed as a control for background signal levels.

[0057] Indicator bacteriophage As described in more detail herein, the compositions, methods, systems and kits of the present invention may include infectious agents for use in the detection of pathogenic microorganisms. In certain embodiments, the present invention includes recombinant indicator bacteriophages, where the bacteriophage genome is genetically modified to include an indicator or reporter gene. In some embodiments, the present invention may include compositions that include recombinant bacteriophages with an indicator gene integrated into the genome of the bacteriophage.

[0058] The recombinant indicator bacteriophage may include a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Late genes code for structural proteins and are therefore generally expressed at higher levels than other phage genes. The late gene region may be a class III gene region and may include a gene for a major capsid protein.

[0059] Some embodiments include designing (and optionally preparing) sequences for homologous recombination downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) sequences for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequences include a codon-optimized reporter gene followed by an untranslated region. The untranslated region may include a phage late gene promoter and a ribosome entry site.

[0060] In some embodiments, the indicator bacteriophage is derived from a Listeria-specific phage. In some embodiments, the selected wild-type bacteriophage or a cocktail of wild-type bacteriophages can infect at least one target Listeria spp. Listeria species are ubiquitous in the environment and are often found in water, sewage, and soil. In some embodiments, the selected wild-type bacteriophage or a cocktail of wild-type bacteriophages can infect one or more, two or more, or three or more target Listeria spp. In certain cases, the target species of Listeria is selected from L. monocytogenes, L. ivanovii, and L. grayi.

[0061] In some embodiments, the selected wild-type bacteriophage is from the Caudovirales order of phages. Caudovirales is an order of tailed bacteriophages with a double-stranded DNA (dsDNA) genome. Each virion of the Caudovirales order has an icosahedral head that contains the virion genome and a flexible tail. The Caudovirales order includes five bacteriophage families: Myoviridae (long contractile tail), Siphoviridae (long non-contractile tail), Podoviridae (short non-contractile tail), Ackermannviridae, and Herelleviridae. The term myovirus can be used to describe any bacteriophage with an icosahedral head and a long contractile tail, including bacteriophages within both the Myoviridae and Herelleviridae families. In some embodiments, the selected wild-type bacteriophage is a member of the Myoviridae family (e.g., Listeria phage B054, Listeria phage LipZ5, Listeria phage PSU-VKH-LP041, and Listeria phage WIL-2). In other embodiments, the selected wild-type bacteriophage is a member of the Herelleviridae family. The genus Pecentumvirus under the family Herelleviridae includes bacteriophages (e.g., Listeria phage LMSP-25, Listeria phage LMTA-148, Listeria phage LMTA-34, Listeria phage LP-048, Listeria phage LP-064, Listeria phage LP-083-2, Listeria phage LP-125, Listeria virus P100, Listeria phage List-36, Listeria phage WIL-1, Listeria phage vB_LmoM_AG20, and Listeria virus A511). LMA4 and LMA8 also appear to be within the genus pecentumvirus under the family Herelleviridae.In other embodiments, the selected wild-type bacteriophage is LMA4 or LMA8. In certain cases, the selected wild-type bacteriophage is LP-ES3A, which is derived from A511 but adapted to infect serotype 3A of Listeria monocytogenes. In yet other embodiments, the selected wild-type bacteriophage is a member of the Ackermannviridae family. In yet other embodiments, the selected wild-type bacteriophage is a member of the Siphoviridae family, including Listeria phages A006, A118, A500, B025, LP-026, LP-030-2, LP-030-3, LP-037, LP-101, LP-110, LP-114, P35, P40, P70, PSA, vB_LmoS_188, and vB_Lmos_293. In another embodiment, the selected wild-type bacteriophage is LP-ES1, which also appears to be in the Homburgvirus genus under the Siphoviridae family.

[0062] In some embodiments, the indicator bacteriophage is derived from a Listeria-specific phage. The indicator bacteriophage is selected from the group consisting of Pecentumvirus, Tequatravirus, ViI, Kuttervirus, Homburgvirus, LMTA-94, LMA4, LMA8, P70, LP-ES1, LP-ES3A, and Listeria phages LMTA-94, P70, T7, T7-like, T4, T4-like, Listeria spp.-specific bacteriophage, ViI, or ViI-like (Kuttervirus, according to GenBank / NCBI) bacteriophage, may be constructed from another bacteriophage having a genome at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the bacteriophage. In other embodiments, the selected wild-type bacteriophage is LP-ES1, LP-ES3A, LMA4, or LMA8. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific for a particular pathogenic microorganism. The genetic modification can avoid the deletion of wild-type genes, and thus the modified phage can remain more similar to the wild-type infectious agent than many commercially available phages. Bacteriophage derived from the environment can be more specific for the bacteria found in the environment, and thus can be genetically distinct from commercially available phages.

[0063] In another aspect of the present invention, the cocktail composition comprises at least one type of recombinant bacteriophage. In some embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A. In other embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA8, LP-ES1, and LP-ES3A.

[0064] Furthermore, phage genes that are considered non-essential may have unrecognized functions. For example, an apparently non-essential gene may have an important function in increasing burst size, such as a clever cutting, fitting, or trimming function in assembly. Therefore, deleting a gene and inserting an indicator may be detrimental. Most phages may package DNA that is a few percent larger than their native genome. In this consideration, a smaller indicator gene may be a more suitable choice for modifying bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (about 500-600 bp to code for), while FLuc is about 62 kDa (about 1,700 bp to code for). Furthermore, the reporter gene should not be endogenously expressed by the bacteria (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. In some embodiments, the indicator gene is luciferase. In other embodiments, the indicator gene is the active subunit of luciferase. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO®, NANOLUC® in combination with an imidazopyrazinon substrate (furimazine) can provide a robust signal with low background.

[0065] In some indicator phage embodiments, the indicator gene may be inserted into a non-translated region to avoid disruption of a functional gene, leaving the wild-type phage gene intact, which may result in greater fitness when infecting non-laboratory strains of bacteria. Additionally, including stop codons in all three reading frames may help increase expression by reducing read-through (also known as leaky expression). This strategy may also eliminate the possibility of low levels of fusion protein being made that appear as a background signal (e.g., luciferase) that cannot be separated from the phage.

[0066] The indicator gene may express various biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases may be used. In alternative embodiments, and as described in more detail herein, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®.

[0067] Thus, in some embodiments, the invention includes a genetically modified bacteriophage that includes a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. Using a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels like viral capsid proteins, but is also not silenced like endogenous bacterial genes or even early viral genes.

[0068] In some embodiments, the late promoter is a Pecentumvirus, Tequatravirus, Homburgvirus, or Kuttervirus promoter, or another phage promoter similar to that found in the selected wild-type phage (i.e., without genetic modification). The late gene region may be a class III gene region and the bacteriophage may be derived from Listeria phages LMTA-94, P70, A511, LP-ES1, LP-ES3A, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, T4-like, ViI, Listeria spp.-specific bacteriophage, or another wild-type bacteriophage having a genome with at least 70%, 75%, 80%, 85%, 90% or 95% homology to LMTA-94, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, ViI, or Listeria-specific bacteriophage. The Pecentumvirus late gene promoter is derived from the T4 or Tequatravirus promoter, since it not only consists of the -10 region, but also the -35 region. This -35 region differs from the standard -35 region found in most bacterial promoters.

[0069] The genetic modification to the infectious agent may include the insertion, deletion, or replacement of a small fragment of nucleic acid, a substantial portion of a gene, or an entire gene. In some embodiments, the inserted or replaced nucleic acid comprises a non-native sequence. The non-native indicator gene may be inserted into the bacteriophage genome such that it is under the control of a bacteriophage promoter. Thus, in some embodiments, the non-native indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification may be configured such that the indicator protein product does not contain the polypeptide of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present invention includes a method for detecting a bacterium of interest, the method including incubating a test sample with such a recombinant bacteriophage.

[0070] In some embodiments, expression of the indicator gene in the progeny bacteriophage after infection of the host bacterium results in a free soluble protein product. In some embodiments, the non-native indicator gene is not linked to a gene encoding a phage structural protein and therefore does not result in a fusion protein. Unlike systems that use a fusion of a detection moiety to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., a soluble luciferase). In some embodiments, the indicator or reporter ideally does not include the bacteriophage structure. That is, the indicator or reporter is not bound to the phage structure. Thus, the indicator or reporter gene is not fused to other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (to a single bacterium) and simplify the assay, allowing the assay to be completed in two hours or less for some embodiments, as opposed to the several hours due to the additional purification steps required for constructs that generate detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding constraints that may alter the conformation of the enzyme active site or access to substrates. If the concentration is 1,000 bacterial cells / mL sample, for example, less than 4 hours post-infection may be sufficient to detect the target bacteria.

[0071] Furthermore, fusion proteins by definition limit the number of moieties attached to a protein subunit in the bacteriophage. For example, the use of a commercially available system designed to act as a platform for fusion proteins results in approximately 415 copies of the fusion moiety in each T7 bacteriophage particle, corresponding to approximately 415 copies of the gene 10B capsid protein. Without this constraint, infected bacteria would be expected to express more copies of the detection moiety (e.g., luciferase) than can fit on the bacteriophage. Furthermore, large fusion proteins (e.g., capsid-luciferase fusions) may inhibit assembly of the bacteriophage particle, thus resulting in fewer bacteriophage progeny. Thus, a soluble, non-fused indicator gene product may be preferred.

[0072] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0073] In some embodiments, the use of soluble detection moieties eliminates the need to remove contaminating parent phage from the lysate of the infected sample cells. With a fusion protein system, any bacteriophage used to infect sample cells has a detection moiety attached and is indistinguishable from daughter bacteriophage that also contain the detection moiety. Because detection of sample bacteria relies on detection of newly created (de novo synthesized) detection moieties, the use of fusion constructs requires an additional step to separate the old (parent) moiety from the newly created (daughter bacteriophage) moiety. This can be accomplished by washing the infected cells multiple times prior to completion of the bacteriophage life cycle, inactivating excess parent phage after infection by physical or chemical means, and / or chemically modifying the parent bacteriophage with a binding moiety (e.g., biotin) that can then be bound and separated (e.g., by streptavidin-coated sepharose beads). However, even with all these attempts at removal, parent phage can persist when high concentrations of parent phage are used to ensure infection of a small number of sample cells, creating a background signal that can obscure detection of signal from progeny phage in infected cells.

[0074] In contrast, with the soluble detection moieties expressed in some embodiments of the invention, purification of the parent phage from the final lysate is not necessary because the parent phage does not have any detection moieties attached. Thus, any detection moieties present after infection must be newly produced, indicating the presence of an infected bacterium or bacteria. To take advantage of this benefit, the production and preparation of parent phage can include purification of the phage from any free detection moieties produced during the production of the parent bacteriophage in the bacterial culture. Standard bacteriophage purification techniques may be used to purify some embodiments of phage in accordance with the invention (e.g., sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, and dialysis or derived techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation may be used as part of the preparation of recombinant phage of the invention to separate parent phage particles from contaminating luciferase protein produced during propagation of phage in the bacterial host. In this way, parent recombinant bacteriophage of the invention are substantially free of any luciferase produced during production in the bacteria. Removal of residual luciferase present in the phage stock may substantially reduce the background signal observed when the recombinant bacteriophage is incubated with a test sample.

[0075] In some embodiments of the modified bacteriophage, the late promoter (class III promoter, e.g., from Pecentumvirus, Homburgvirus, T7, T4, ViI, or LMA4 / 8) has high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes for the structural proteins that are assembled into bacteriophage particles. These proteins are the most abundant proteins made by the phage, since each bacteriophage particle contains tens or hundreds of copies of these molecules. The use of viral late promoters can optimally ensure high levels of expression of the luciferase detection moiety. The use of late viral promoters derived from wild-type bacteriophages under which the indicator phage is specific or active (e.g., the indicator phage derived from Pecentumvirus, Homburgvirus, T4, T7, ViI, or LMA4 / 8 late promoters and Pecentumvirus, T4, T7-, ViI-, or LMA-based systems) can further ensure optimal expression of the detection moiety. The use of standard bacterial (non-viral / non-bacteriophage) promoters can in some cases be detrimental to expression, since these promoters are often downregulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production). Thus, in some embodiments, the phage is engineered to encode and express high levels of a soluble (free) indicator moiety, preferably with an arrangement in the genome that does not limit expression to the number of subunits of the phage structural components.

[0076] The compositions of the invention may include one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may include a cocktail of different indicator phages that may encode and express the same or different indicator proteins. In some embodiments, the cocktail of bacteriophages includes at least two different types of recombinant bacteriophages.

[0077] Method for preparing indicator bacteriophage An embodiment of the method for making indicator bacteriophages begins with the selection of a wild-type bacteriophage for genetic modification. Some bacteriophages are highly specific for the target bacteria. This provides an opportunity for highly specific detection.

[0078] Thus, the methods of the present invention take advantage of the high specificity of binding agents associated with infectious agents that recognize and bind to specific microorganisms of interest as a means to amplify the signal and thereby detect low levels of microorganisms (e.g., single microorganisms) present in a sample. For example, infectious agents (e.g., bacteriophages) specifically recognize surface receptors of specific microorganisms and therefore specifically infect those microorganisms. Thus, these infectious agents can be suitable binding agents to target microorganisms of interest.

[0079] Some embodiments of the present invention take advantage of the binding specificity and high level gene expression capacity of recombinant bacteriophage for rapid and sensitive targeting to infect and facilitate detection of bacteria of interest. In some embodiments, Listeria-specific bacteriophage are genetically modified to contain a reporter gene. In some embodiments, the late gene region of the bacteriophage is genetically modified to contain a reporter gene. In some embodiments, the reporter gene is located downstream of the major capsid gene. In other embodiments, the reporter gene is located upstream of the major capsid gene. In some embodiments, the inserted gene construct further comprises a dedicated promoter, itself exogenous, to drive expression of the indicator gene. The exogenous promoter is in addition to any endogenous promoters in the phage genome. Because bacteriophage generate polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron in the transcript. Conventional recombinant constructs only use endogenous bacteriophage promoters to drive inserted genes. In contrast, adding an additional promoter upstream of the reporter gene and ribosome binding site can increase gene expression by acting as a second start site for transcription. The complex and compact genomes of viruses often have overlapping genes in different frames, sometimes in two different orientations.

[0080] Some embodiments of the method for preparing a recombinant indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, such as Listeria spp., preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid / vector and the bacteriophage genome, and isolating a specific clone of the recombinant bacteriophage.

[0081] Various methods are known for designing and preparing homologous recombination plasmids.Various methods are known for transforming bacteria with plasmids, including heat shock, F pilus-mediated bacterial conjugation, electroporation and other methods.Various methods are also known for isolating specific clones after homologous recombination.Some method embodiments described herein utilize specific strategies.

[0082] Thus, some embodiments of the method for preparing an indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, determining the native sequence in the late region of the genome of the selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing sequences for homologous recombination adjacent to the major capsid protein gene, where the sequences comprise a codon-optimized reporter gene, incorporating the sequences designed for homologous recombination into a plasmid / vector, transforming the plasmid / vector into the target pathogenic bacterium, selecting for the transformed bacteria, infecting the transformed bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid and the bacteriophage genome, determining the titer of the resulting recombinant bacteriophage lysate, and performing a limiting dilution assay to enrich and isolate the recombinant bacteriophage. Some embodiments further include repeating the limiting dilution and titering steps as necessary after the first limiting dilution assay until recombinant bacteriophage represent a detectable proportion of the mixture. For example, in some embodiments, the limiting dilution and titering steps may be repeated until at least 1 / 30 of the bacteriophage in the mixture are recombinant before isolating a specific clone of recombinant bacteriophage. A 1:30 recombinant:wild type ratio is expected to yield an average of 3.2 transducing units (TU) per 96 plaques (e.g., in a 96-well plate) in some embodiments. The initial ratio of recombinant phage:wild type phage may be determined by performing a limiting dilution assay based on TCID50 (tissue culture infectious dose 50%) as previously described in U.S. Patent Application Serial No. 15 / 409,258. By Poisson distribution, a 1:30 ratio results in a 96% chance of observing at least 1 TU anywhere in the 96 wells.

[0083] FIG. 1 shows a schematic representation of the genomic structure of a recombinant indicator bacteriophage of the present invention. For the embodiment shown in FIG. 1, the detection moiety is encoded by a luciferase gene 100 inserted into a late (class III) gene 110 that is expressed late in the viral life cycle. Late genes are generally expressed at higher levels than other phage genes because they code for structural proteins. Thus, in the embodiment of the recombinant phage shown in FIG. 1, the indicator gene (i.e., luciferase) is inserted into the late gene region immediately following the gene 120 of the major capsid protein (cps), a construct that includes the luciferase gene 100. In some embodiments, the construct shown in FIG. 1 may include stop codons in all three reading frames to ensure that luciferase is not incorporated into the cps gene product via the creation of a fusion protein. As also shown in FIG. 1, the construct may include an additional dedicated late promoter 130 to drive transcription and expression of the luciferase gene. The construct also contains a ribosome binding site (RBS) 140. This construct ensures that soluble luciferase is produced such that expression is restricted to the number of unique capsid proteins in the phage display system.

[0084] As noted herein, in certain embodiments, it may be preferable to utilize infectious agents that have been isolated from the environment for production of the infectious agents of the present invention. In this manner, naturally derived microbial specific infectious agents may be produced.

[0085] For example, Figure 2 shows the genome of bacteriophage LMA4, a wild-type bacteriophage that specifically infects Listeria spp. As discussed in the Examples, the major capsid protein (cps) 240 and various other structural genes are within the late gene region 210, which consists of structural genes and codes for virion proteins. A gene 220 encoding a tRNA shows genomic sequence adjacent to, but outside, the late gene region. A hypothetical gene homologous to the putative prohead protease of Listeria phage LMTA-94 230 is upstream of cps 240, which consists of structural genes and codes for virion proteins. Other late genes shown are a homolog to the putative major capsid protein (cps) 240 of Listeria phage LMTA-94, followed by a transcription terminator 250, and a homolog to Listeria phage LMTA-94 tail sheath protein (tsh) 260. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, so long as the late gene promoter and / or other similar control elements are used.

[0086] There are many known methods and commercial products for preparing plasmids. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning and other techniques can be used in combination to prepare plasmids. Synthetic plasmids can also be ordered commercially (e.g., GeneWiz). Cosmids can also be used to selectively edit the bacteriophage genome, or the CRISPR / CAS9 system can also be used. Some embodiments of the method for preparing recombinant indicator bacteriophage include designing a plasmid that can easily recombine with a wild-type bacteriophage genome to generate a recombinant genome. In designing the plasmid, some embodiments include the addition of a codon-optimized reporter gene, such as a luciferase gene. Some embodiments further include the addition of elements to the upstream untranslated region. For example, in designing a plasmid for recombination with a Listeria-specific bacteriophage genome, an upstream untranslated region can be added between the sequence encoding the C-terminus of the gp23 / major capsid protein and the start codon of the NANOLUC® reporter gene. The untranslated region may contain a promoter (e.g., T4, Tequatravirus, Homburgvirus, T7, T7-like, Pecentumvirus, Listeria-specific bacteriophage, ViI, or Kuttervirus promoter). The untranslated region may also contain a ribosome entry / binding site (RBS), also known as the "Shine-Dalgarno sequence" in bacterial systems. Either or both of these elements, or other untranslated elements, may be embedded within a short upstream untranslated region made of random sequences containing approximately the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence, since that would act as the start codon.

[0087] The compositions of the invention may include various infectious agents and / or indicator genes. For example, Figure 3 shows the homologous recombination plasmid constructs used in making the indicator phages specific for Listeria spp. Constructs were made and used in recombination with Listeria spp. phage LMA4, Listeria spp. phage LMA8, Listeria spp. phage LP-ES3A, Listeria spp. phage LP-ES1 or other Listeria-specific phages to generate the recombinant bacteriophages of the invention. The constructs in FIG. 3 show general schematics of the recombinant plasmids used for homologous recombination insertion of NANOLUC® luciferase into both Listeria spp. Pecentumvirus phages LMA4 and LMA8 (with 500 bp of upstream and downstream homologous sequences, respectively): the homologous recombination plasmid pCE104.HR.ListeriaPhage.NANOLUC.v2. Pecentumvirus.NANOLUC.v2, and the recombinant plasmid used for homologous recombination insertion of NANOLUC® luciferase into Listeria spp. Homburgvirus phage LP-ES1, pCE104.HR.LP-ES1.NanoLuc.

[0088] In certain embodiments, the plasmid is designated pCE104.HR.Pecentumvirus.NanoLuc.v2. The detection / indicator portion is encoded by the NANOLUC® reporter gene 300. The insert (represented by a series of boxes) is in a Gram-positive shuttle vector, pCE104 310. The upstream homologous recombination region consists of 500 bp 320 of the major capsid protein C-terminal fragment. The Pecentumvirus late promoter consensus sequence and Shine-Dalgarno ribosome entry / binding site are within the 5' untranslated region 330. The codon-optimized NANOLUC® reporter gene 300 immediately follows. The endogenous transcription terminator appears next, along with the untranslated region (UTR) and hypothetical protein N-terminal fragment, consisting of the downstream homologous recombination 340, at the end of the homologous recombination region.

[0089] The major capsid protein fragment is part of a structural gene that encodes a virion protein, which is expressed at very high levels, so any gene inserted into this region can be expected to have a similar expression level, provided the late gene promoter and / or other similar control elements are used.

[0090] In some embodiments, the indicator phage according to the invention comprises a Listeria-specific bacteriophage genetically engineered to contain a reporter gene (e.g., a luciferase gene). For example, the indicator phage can be a Listeria spp.-specific bacteriophage whose genome contains the sequence of the NANOLUC® gene. The recombinant Listeria-specific NanoLuc bacteriophage genome can further comprise a consensus promoter or another late promoter of Pecentumvirus, T4, T7, Listeria-specific, ViI, LMA4 or LMA8 bacteriophage. In a further embodiment, the promoter is an exogenous promoter. Insertion of an exogenous promoter to drive expression of the indicator gene is advantageous in that expression is limited by the expression of other phage proteins (e.g., major capsid protein).

[0091] Thus, in an embodiment of the recombinant phage produced as a result of recombination, the indicator gene (i.e., NANOLUC®) is inserted into the late gene region immediately downstream of the gene encoding the major capsid protein, thus creating a recombinant bacteriophage genome containing the NANOLUC® gene. The construct further comprises the consensus promoter of Listeria phage LMTA-94, T4, T7, Listeria specific bacteriophage, ViI, or another late promoter or another suitable promoter to drive transcription and expression of the luciferase gene. The construct may also include a composite untranslated region synthesized from several UTRs. This construct allows for the production of a soluble luciferase such that expression is unique to the phage display system. This ensures that there is no limit to the number of peptide proteins.

[0092] Recombinant phage generated by homologous recombination of a plasmid designed for recombination with a wild-type phage genome can be isolated from a mixture containing a very small percentage (e.g., 0.005%) of the total phage genome. After isolation, large-scale production can be performed to obtain high-titer recombinant indicator phage stocks suitable for use in Listeria spp. detection assays. In addition, cesium chloride isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein to reduce background.

[0093] Methods using infectious agents to detect Listeria spp. As noted herein, in certain embodiments, the invention may include methods for using infectious particles to detect microorganisms. The methods of the invention may be embodied in a variety of ways.

[0094] In one embodiment, the invention may include a method for detecting a bacterium of interest in a sample, comprising incubating the sample with a bacteriophage that infects the bacterium of interest, where the bacteriophage contains an indicator gene, such that expression of the indicator gene during bacteriophage replication following infection of the bacterium of interest results in a soluble indicator protein product, and detecting the indicator protein product, where positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample. In certain cases, the invention may include a method for detecting Listeria spp. in a sample, comprising incubating the sample with a cocktail composition comprising at least one Listeria-specific recombinant bacteriophage; and detecting an indicator protein product produced by the recombinant bacteriophage, where positive detection of the indicator protein product indicates that Listeria spp. is present in the sample.

[0095] In some embodiments, at least one type of recombinant bacteriophage is constructed from LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A, hi other embodiments, at least one type of recombinant bacteriophage is constructed from LMA8, LP-ES1, and LP-ES3A.

[0096] In certain embodiments, assays may be performed utilizing a general concept that may be modified to accommodate different sample types or sizes and assay formats. Embodiments using the recombinant bacteriophages of the invention (i.e., indicator bacteriophages) may be performed in the following time periods depending on the sample type, sample size, and assay format: 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, 5.0 hours, 5.5 hours, 6.0 hours, 6.5 hours, 7.0 hours, 7.5 hours, 8.0 hours, 8.5 hours, 9.0 hours, 9.5 hours, 10.0 hours, 10.5 hours, 11.0 hours, 11.5 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 105 hours In some embodiments, the assay may allow for rapid detection of specific bacterial strains such as Listeria spp. in total assay times of less than 12.5 hours, 13.0 hours, 13.5 hours, 14.0 hours, 14.5 hours, 15.0 hours, 15.5 hours, 16.0 hours, 16.5 hours, 17.0 hours, 17.5 hours, 18.0 hours, 18.5 hours, 19.0 hours, 19.5 hours, 20.0 hours, 21.0 hours, 21.5 hours, 22.0 hours, 22.5 hours, 23.0 hours, 23.5 hours, 24.0 hours, 24.5 hours, 25.0 hours, 25.5 hours or 26.0 hours. For example, the amount of time required may be somewhat shorter or longer depending on the strain of bacteriophage and the strain of bacteria being detected in the assay, the type and size of the sample being tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target contaminating bacteria.

[0097] The bacteriophage (e.g., T7, T4, P70, P100, A511, LP-ES3A, LP-ES1, LMA4 or LMA8 phage) can be engineered to express soluble luciferase during phage replication. Expression of luciferase is driven by a viral capsid promoter (e.g., the bacteriophage Pecentumvirus or T4 late promoter), resulting in high expression. Because parent phages are prepared such that they do not contain luciferase, any luciferase detected in the assay must come from replicating progeny phage during infection of bacterial cells. Thus, there is generally no need to separate the parent phage from the progeny phage.

[0098] 4 illustrates a filter plate assay for detecting Listeria using modified bacteriophage according to one embodiment of the present invention. Briefly, a sample 416 containing bacteria of interest 418 is added to a well 402 of a multi-well filter plate 404 and concentrated by centrifugation 406 to remove liquid from the sample. A genetically modified phage 420 is added to the well and incubated with additional media for a period of time sufficient for adsorption 408, followed by infection of the target bacteria and the phage life cycle to proceed 410 (e.g., about 240 minutes). Finally, a luciferase substrate is added and reacts with any luciferase present 424. The resulting luminescence is measured 414 with a luminometer that detects luciferase activity 426.

[0099] In some embodiments, enrichment of bacteria in the sample is not required before testing. In some embodiments, the sample can be enriched before testing by incubation in conditions that promote growth. In such embodiments, the enrichment period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours or longer, depending on the type and size of the sample.

[0100] In some embodiments, the indicator bacteriophage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Thus, the method can include detecting the indicator moiety generated during phage replication, where detection of the indicator indicates that the bacterium of interest is present in the sample.

[0101] In one embodiment, the invention may include a method for detecting a bacterium of interest in a sample comprising incubating the sample with a recombinant bacteriophage that infects the bacteriophage, wherein the recombinant bacteriophage contains an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication following infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of the bacterium of interest present in the sample.

[0102] As described in more detail herein, the methods and systems of the present invention can utilize a range of concentrations of parent indicator bacteriophage to infect bacteria present in a sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria present in very low numbers in the sample, such as 10 cells. In some embodiments, the phage concentration can be sufficient to find, bind to, and infect target bacteria in less than one hour. In other embodiments, these events can occur in less than two hours, or less than three hours, or less than four hours after addition of indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubating step is 1×10 5 PFU / mL or higher than 1 × 10 6 PFU / mL or greater than 1 × 10 7 Higher than PFU / mL.

[0103] In certain embodiments, the recombinant infectious agent may be purified to be free of any residual indicator protein that may be generated during production of the infectious agent stock. Thus, in certain embodiments, the recombinant bacteriophage may be purified using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. If the infectious agent is a bacteriophage, this purification may have the added benefit of removing bacteriophage that do not have DNA (i.e., empty phage or "ghosts").

[0104] In some embodiments of the method of the present invention, the microorganism can be detected without any isolation or purification of the microorganism from the sample.For example, in certain embodiments, a sample containing one or several target microorganisms can be directly applied to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is carried out in the assay vessel.Various embodiments of such assays are disclosed herein.

[0105] Test sample aliquots can be dispensed directly into wells of a multi-well plate, indicator phage can be added, and after a sufficient period for infection, lysis buffer can be added as well as a substrate for the indicator moiety (e.g., luciferase substrate for a luciferase indicator) and assayed for detection of the indicator signal. Some embodiments of the method can be performed on a filter plate. Some embodiments of the method can be performed with or without concentration of the sample prior to infection with indicator phage.

[0106] For example, in many embodiments, a multi-well plate is used to perform the assay. The choice of plate (or any other container in which the detecting step can be performed) can affect the detecting step. For example, some plates can include a colored or white background, which can affect the detection of light emission. Generally, white plates have higher sensitivity, but also produce higher background signals. Other colors of plates can produce lower background signals, but have slightly lower sensitivity. Furthermore, one reason for background signals is light leakage from one well to another adjacent well. There are some plates with white wells, but the rest of the plate is black. This allows for high signals inside the wells, but prevents light leakage from well to well, thus reducing background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal for the assay.

[0107] The methods of the invention may include various other steps to increase sensitivity. For example, as discussed in more detail herein, the methods may include washing the captured and infected bacteria after adding the bacteriophage but before incubation to remove excess parent bacteriophage and / or luciferase or other reporter proteins that contaminate the bacteriophage preparation.

[0108] In some embodiments, detection of the microorganism of interest may be completed without the need to culture the sample as a method to increase the population of the microorganism. For example, in certain embodiments, the total time required for detection is less than 28.0 hours, 27.0 hours, 26.0 hours, 25.0 hours, 24.0 hours, 23.0 hours, 22.0 hours, 21.0 hours, 20.0 hours, 19.0 hours, 18.0 hours, 17.0 hours, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, or 1.0 hours. Minimizing the time to results is critical in food and environmental testing for pathogens.

[0109] In contrast to assays known in the art, the methods of the present invention can detect individual microorganisms. Thus, in certain embodiments, the methods can detect as few as 10 cells of the microorganism present in a sample. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Listeria spp. In one embodiment, the recombinant bacteriophage can distinguish Listeria spp. in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the recombinant bacteriophage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample.

[0110] Thus, aspects of the invention provide methods for the detection of a microorganism in a test sample via an indicator moiety. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety may be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator moiety may react with a substrate to emit a detectable signal or may emit an intrinsic signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity may reveal the presence of as few as 50, 40, 30, 20, 10, 5, or 2 cells of the microorganism of interest in a test sample. In some embodiments, as little as one cell of the microorganism of interest may produce a detectable signal. In some embodiments, the bacteriophage is a Pecentumvirus, Tequatravirus, Homburgvirus, or Kuttervirus bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Listeria-specific bacteriophage. In certain embodiments, the recombinant Listeria-specific bacteriophage is highly specific for Listeria spp.

[0111] In some embodiments, the indicator moiety encoded by the infectious agent may be detectable during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator moieties are known in the art, and many are commercially available. In some embodiments, the indicator phage contains an enzyme, which serves as the indicator moiety. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may emit light and / or be detectable by a color change in a converted substrate. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal can also be suitable indicator moieties.

[0112] Thus, in some embodiments, the recombinant bacteriophage of the method, system or kit is prepared from a wild-type Listeria-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein, etc.). The indicator may emit light and / or be detectable by a color change. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of Oplophorus luciferase, firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase, or an engineered luciferase, such as NANOLUC®, Rluc8.6-535, or orange nano-lantern.

[0113] Detecting the indicator may include detecting light emission. In some embodiments, a luminometer may be used to detect the reaction of the indicator (e.g., luciferase) with the substrate. Detection of RLU may be achieved with a luminometer, or other machines or devices may also be used. For example, a spectrophotometer, CCD camera, or CMOS camera may detect color changes and other light emissions. Although absolute RLU is important for detection, a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is also required to ensure that single cells or a small number of cells are detected.

[0114] In some embodiments, the indicator phage is engineered to contain a gene for an enzyme (e.g., luciferase) that is produced only upon infection of the bacterium that the phage specifically recognizes and infects. In some embodiments, the indicator moiety is expressed late in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number.

[0115] Thus, in some embodiments utilizing indicator phages, the present invention provides: The present invention includes a method for detecting a microorganism of interest, comprising the steps of capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phages; allowing time for infection and replication to produce progeny phage and express a soluble indicator moiety; and detecting the progeny phage, preferably the indicator, wherein detection of the indicator indicates that the bacterium is present in the sample.

[0116] For example, in some embodiments, test sample bacteria may be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a 0.45 μm pore size spin filter or plate filter). In one embodiment, the infectious agent (e.g., indicator phage) is added in a minimal volume to the sample captured directly on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are then washed one or more times to remove excess unbound infectious agents. In an embodiment, medium (e.g., Luria-Bertani (LB) broth, buffered peptone water (BPW) or Tryptic Soy broth or Tryptone Soy broth (TSB), brain heart infusion (BHI), buffered Listeria enrichment broth (BLEB) University of Vermont (UVM) broth, or Fraser broth) may be added for an additional incubation time to allow bacterial cell and phage replication and high level expression of the gene encoding the indicator moiety. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage only needs to be long enough for a single phage life cycle. The amplifying power of using bacteriophage was previously thought to require more time as the phage replicates over several cycles. A single replication cycle of the indicator phage can be sufficient to facilitate sensitive and rapid detection in accordance with some embodiments of the present invention.

[0117] In some embodiments, an aliquot of a test sample containing bacteria may be applied to a spin column, and after infection with recombinant bacteriophage and optional washing to remove any excess bacteriophage, the amount of soluble indicator detected is proportional to the amount of bacteriophage produced by the infected bacteria.

[0118] Soluble indicators (e.g., luciferase) released into the surrounding liquid upon lysis of the bacteria can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter and the filtrate is collected in a new container for assay (e.g., in a luminometer) after which a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0119] In various embodiments, the purified parent indicator phage does not contain the detectable indicator itself, since the parent phage may be purified before it is used to incubate with a test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the invention, the parent phage may be purified to eliminate any present indicator protein (e.g., luciferase). In some embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. Thus, in many embodiments, it is not necessary to separate the parent phage from the progeny phage prior to the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a bacteriophage. In one embodiment, the indicator moiety is a soluble luciferase, which is released upon lysis of the host microorganism.

[0120] Thus, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) may be incubated with the portion of the sample that remains on the filter or that remains bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a regular 96-well plate) and the substrate reaction may be detected by placing the plate directly into the luminometer.

[0121] For example, in one embodiment, the invention may include a method for detecting Listeria spp., comprising the steps of infecting cells captured on a 96-well filter plate with multiple parent indicator phages capable of expressing luciferase upon infection; washing away excess phages; adding BHI broth and allowing time for phages to replicate and lyse the specific Listeria spp. targets (e.g., 60-240 minutes); and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, where detection of luciferase activity indicates that Listeria spp. is present in the sample.

[0122] In another embodiment, the invention may include a method for detecting Listeria spp., comprising the steps of infecting cells in a liquid solution or suspension in a 96-well plate with a plurality of parent indicator phages capable of expressing luciferase upon infection; allowing time for the phages to replicate and lyse the specific Listeria spp. target (e.g., 60-240 minutes); and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, where detection of luciferase activity indicates that Listeria spp. is present in the sample. In such an embodiment, the capturing step is not required. In some embodiments, the liquid solution or suspension may be a consumable test sample (e.g., vegetable wash). In some embodiments, the liquid solution or suspension may be concentrated Luria-Bertani (LB) broth, buffered peptone water (BPW) or vegetable washes enriched with Tryptic Soy Broth (TSB) or Tryptone Soy Broth, Brain Heart Infusion (BHI), buffered Listeria enriched broth (BLEB), University of Vermont (UVM) broth, or Fraser broth. In some embodiments, the liquid solution or suspension may be bacteria diluted in BHI broth.

[0123] In some embodiments, lysis of the bacteria may occur before, during, or after the detection step. Experiments suggest that infected, unlysed cells may be detectable in some embodiments upon addition of luciferase substrate. Possibly, luciferase may leave the cell and / or luciferase substrate may enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required for detection if only luciferase released into the lysate (and luciferase is not even in intact bacteria) is analyzed in the luminometer. However, for embodiments utilizing a filter plate or 96-well plate with a solution or suspension sample, lysis is not essential for detection if the original plate filled with intact and lysed cells is directly assayed in the luminometer.

[0124] In some embodiments, the reaction of the indicator moiety (e.g., luciferase) with the substrate may continue for 60 minutes or longer, and detection at various time points may be desirable to optimize sensitivity. For example, in an embodiment using a 96-well filter plate as the solid support and luciferase as the indicator, luminometer readings may be taken initially and at 10 or 15 minute intervals until the reaction is complete.

[0125] Surprisingly, the high concentrations of phage utilized to infect the test sample successfully achieved detection of very small numbers of target microorganisms in a very short time frame. Incubating the phage with the test sample, in some embodiments, only needs to be long enough for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubation step is greater than 7×10 6 , 8×10 6 , 9×10 6 , 1.0×10 7 , 1.1×10 7 , 1.2×107 , 1.3×10 7 , 1.4×10 7 , 1.5×10 7 , 1.6×10 7 , 1.7×10 7 , 1.8×10 7 , 1.9×10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 , or 1.0×10 8 Higher than PFU / mL.

[0126] The success of such high concentrations of phage is surprising because many of the phage were previously associated with "non-infectious lysates" that killed target cells and thereby prevented the generation of useful signals from earlier phage assays. Clean-up of prepared phage stocks as described herein is believed to help alleviate this problem (e.g., clarification by cesium chloride isopycnic density gradient ultracentrifugation), because in addition to removing any contaminating luciferase associated with the phage, this clean-up may also remove ghost particles (particles that have lost DNA). The ghost particles may lyse bacterial cells via "non-infectious lysates," prematurely killing the cells and thereby preventing the generation of indicator signals. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride clean-up) may have more than 50% ghosts. These ghost particles may contribute to the early death of the microorganism through the action of many phage particles punching holes in the cell membrane. Thus, ghost particles may have contributed to previous problems where high PFU concentrations were reported to be harmful. Furthermore, the very clean phage preparation allows the assay to be performed without a washing step, which allows the assay to be performed without an initial enrichment step. Some embodiments include an initial enrichment step, which in some embodiments allows for shorter enrichment incubation times.

[0127] Some embodiments of the test method may further include a confirmatory assay. A variety of assays are known in the art to confirm the initial results, usually at a later time point. For example, the sample may be cultured (e.g., selective chromogenic plating), PCR may be utilized to confirm the presence of microbial DNA, or other confirmatory assays may be used to confirm the initial results.

[0128] In certain embodiments, the methods of the invention may combine the use of binding agents (e.g., antibodies) to purify and / or concentrate a microorganism of interest (e.g., Listeria spp.) from the sample in addition to detection with an infectious agent. For example, in certain embodiments, the invention encompasses a method for detecting a microorganism of interest in a sample, comprising capturing the microorganism from the sample onto a support using a capture antibody specific for the microorganism of interest (e.g., Listeria spp.); incubating the sample with a recombinant bacteriophage that infects Listeria spp., where the recombinant bacteriophage contains an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product; and detecting the indicator protein product, where positive detection of the indicator protein product indicates that Listeria spp. is present in the sample.

[0129] In some embodiments, synthetic phages are designed to optimize desired traits for use in pathogen detection assays. In some embodiments, bioinformatics and previous analysis of genetic modifications are used to optimize desired traits. For example, in some embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to entire genuses of bacteria, or groups of specific species within a genus. In this way, the phage can be optimized to detect broader or narrower groups of pathogens. In some embodiments, the synthetic phage can be designed to improve expression of the reporter gene. Additionally and / or alternatively, in some cases, the synthetic phage can be designed to increase the burst size of the phage to improve detection.

[0130] In some embodiments, the stability of the phage may be optimized to improve shelf life. For example, enzybiotic solubility may be increased to increase subsequent phage stability. Additionally and / or alternatively, phage thermostability may be optimized. Thermostable phage better preserve functional activity during storage, thereby increasing shelf life. Thus, in some embodiments, the thermostability and / or pH tolerance may be optimized.

[0131] In some embodiments, the genetically modified phage or the synthetically derived phage comprises a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the phage genome comprises an indicator gene (e.g., a luciferase gene or another gene encoding a detectable indicator).

[0132] Systems and kits of the present invention In some embodiments, the present invention includes a system (e.g., an automated system or kit) that includes components for carrying out the methods disclosed herein. In some embodiments, the system or kit according to the present invention includes an indicator phage. The methods described herein may also utilize such an indicator phage system or kit. Some embodiments described herein are particularly suitable for automation or kits, given the minimal amount of reagents and materials required to carry out the methods. In certain embodiments, each of the components of the kit may include a self-contained unit that is deliverable from a first site to a second site.

[0133] In some embodiments, the present invention includes a system or kit for rapid detection of a microorganism of interest in a sample. The system or kit may, in certain embodiments, include a component for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety, and a component for detecting the indicator moiety. In some embodiments of both the systems and kits of the present invention, the infectious agent is a recombinant bacteriophage that infects the bacteriophage of interest, and the recombinant bacteriophage includes an indicator gene inserted into the late gene region of the bacteriophage as the indicator moiety, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. Some systems further include a component for capturing the microorganism of interest on a solid support.

[0134] In other embodiments, the invention includes a method, system, or kit for rapid detection of a microorganism of interest in a sample, comprising an infectious agent component specific for the microorganism of interest, wherein the infectious agent comprises an indicator moiety and a component for detecting the indicator moiety. In some embodiments, the bacteriophage is a Tequatravirus, ViI, Kuttervirus, Homburgvirus, Pecentumvirus, or Listeria spp. specific bacteriophage. In one embodiment, the recombinant bacteriophage is derived from a Listeria spp. specific bacteriophage. In certain embodiments, the recombinant bacteriophage is highly specific for a particular bacterium. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Listeria spp. In embodiments, the recombinant bacteriophage can distinguish Listeria spp. in the presence of other types of bacteria. In certain embodiments, the system or kit detects as few as 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 specific bacteria in the sample.

[0135] In certain embodiments, the system and / or kit may further include a component for washing the captured microbial sample. Additionally or alternatively, the system and / or kit may further include a component for determining the amount of the indicator moiety, where the amount of indicator moiety detected corresponds to the amount of microorganisms in the sample. For example, in certain embodiments, the system or kit may include a luminometer or other device for measuring luciferase enzyme activity.

[0136] In some systems and / or kits, the same components may be used for multiple steps, in some systems and / or kits, the steps are automated or controlled by a user via computer input and / or a liquid handling robot performs at least one step.

[0137] Thus, in certain embodiments, the present invention may include a system or kit for rapid detection of a microorganism of interest in a sample, the system or kit including a component for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microbial sample to remove unbound infectious agents; and a component for detecting the indicator moiety. In some embodiments, the same component may be used for the capturing and / or incubating and / or washing steps (e.g., a filter component). Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, where the amount of indicator moiety detected corresponds to the amount of the microorganism in the sample. Such a system may include various embodiments and subembodiments similar to those described above with respect to the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage. In a computerized system, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination of these).

[0138] In some embodiments, the system may include components for isolating the microorganism of interest from other components in the sample.

[0139] In one embodiment, the invention encompasses a system or kit that includes components for detecting a microorganism of interest, the system including a component for isolating at least one microorganism from other components in the sample, a component for infecting the at least one microorganism with a plurality of parent infectious agents, a component for lysing at least one infected microorganism to release progeny infectious agents present in the microorganism, and a component for detecting the progeny infectious agents, or, with greater sensitivity, a component for detecting a soluble protein encoded and expressed by the infectious agent, where the infectious agent or a soluble protein product of the infectious agent indicates the presence of the microorganism in the sample. The infectious agent may include a Listeria-specific NANOLUC® bacteriophage carrying a NANOLUC® indicator gene.

[0140] The system or kit may include various components for detection of progeny infectious agents. For example, in one embodiment, the progeny infectious agent (e.g., bacteriophage) may include an indicator moiety. In one embodiment, the indicator moiety in the progeny infectious agent (e.g., bacteriophage) may be a detectable moiety (e.g., a soluble luciferase protein) that is expressed during replication.

[0141] In other embodiments, the present invention may include a kit for rapid detection of a microorganism of interest in a sample, the system including a component for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microbial sample to remove unbound infectious agents; and a component for detecting the indicator moiety. In some embodiments, the same component may be used for the capturing and / or incubating and / or washing steps. Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, where the amount of indicator moiety detected corresponds to the amount of the microorganism in the sample. Such a kit may include various embodiments and subembodiments similar to those described above with respect to the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0142] In some embodiments, kits may include components for isolating the microorganism of interest from other components in the sample.

[0143] These systems and kits of the invention include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of suitable devices for carrying out the described method. The components need not be integrally connected or mounted relative to each other in any particular manner. The invention includes any suitable arrangement of the components relative to each other. For example, the components need not be in the same space. However, in some embodiments, the components are connected to each other in an integral unit. In some embodiments, the same component may perform multiple functions.

[0144] Computer system and computer readable medium The system may be embodied in the form of a computer system, as described in the present technology or any of its components. Representative examples of computer systems include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps constituting the methods of the present technology.

[0145] The computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include a memory. The memory may include a random access memory (RAM) and a read-only memory (ROM). The computer system may further include a storage device. The storage device may be a hard disk drive or a removable storage device (e.g., a floppy disk drive, an optical disk drive, etc.). The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet through an I / O interface. The communication unit allows the transfer of data to and receipt of data from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks (e.g., LAN, MAN, WAN, and Internet). The computer system may thus facilitate input from a user through input devices accessible to the system via an I / O interface.

[0146] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) that stores instructions that, when executed by a server's processor, cause the computing device to perform its intended functions. Suitable implementations of such operating systems and the general functionality of such computing devices are known or commercially available and are readily implemented by those of skill in the art, especially in light of the disclosure herein.

[0147] The computer system executes a set of instructions stored in one or more memory devices to process input data. The memory devices may also hold data or other information as described. The memory devices may be in the form of information sources or physical memory devices present in a processing machine.

[0148] The environment may include various data stores and other memory and storage media as discussed above. These may reside in various locations (e.g., in storage media local to (and / or residing in) one or more of the computers, or remote from any or all of the computers across a network). In a particular group of embodiments, the information may reside on a storage area network ("SAN") with which those skilled in the art are familiar. Similarly, any necessary files for performing the functions ascribed to the computers, servers, or other network devices may be stored locally and / or remotely, as appropriate. Where the system includes computing devices, each such device may include hardware elements that may be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touch screen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices, such as disk drives, optical storage devices, and solid-state storage devices, such as random access memory ("RAM") or read-only memory ("ROM"), as well as removable media devices, memory cards, flash cards, and the like.

[0149] Such devices may also include computer-readable storage media readers, communication devices (e.g., modems, network cards (wireless or wired), infrared communication devices, etc.), and working memory as described above. The computer-readable storage media readers may be connected to or configured to accept computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The systems and various devices also typically include many software applications, modules, services, or other elements located within at least one working memory device, including operating systems and application programs (e.g., client applications or web browsers). It should be recognized that alternative embodiments may have many variations from those described above. For example, customized hardware may also be used, and / or specific elements may be implemented in hardware, software (including portable software (e.g., applets)), or both. Additionally, connections to other computing devices (e.g., network input / output devices) may be used.

[0150] Non-transitory storage media and computer readable media for containing the code or portions of the code may include any suitable media known or used in the art, including storage media and communication media, including but not limited to volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage and / or transmission of information, such as computer readable instructions, data structures, program modules, or other data, including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by the system device. Based on the above disclosure and teachings provided herein, one skilled in the art will recognize other ways and / or methods for implementing the various embodiments.

[0151] A computer-readable medium may include, but is not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, content addressable memories ("CAMs"), DDRs, flash memories (e.g., NAND Flash or NOR Flash), ASICs, configured processors, optical storage media, magnetic tapes or other magnetic storage media, or any other media from which a computer processor may read instructions. In one embodiment, the computing device may include a single type of computer-readable medium (e.g., random access memory (RAM)). In other embodiments, the computing device may include two or more types of computer-readable media (e.g., random access memory (RAM), disk drives, and caches). The computing device may be in communication with one or more external computer-readable media (e.g., an external hard disk drive, or an external DVD or Blu-ray drive).

[0152] As discussed above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and state machines. Such a processor may further include a programmable electronic device (e.g., a PLC), a programmable interrupt controller (PIC), a programmable logic device (PLD), a programmable read-only memory (PROM), an electronically programmable read-only memory (EPROM or EEPROM), or other similar device.

[0153] The computing device includes a network interface. In some embodiments, the network interface is configured to communicate over a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface may enable communication over a network (e.g., CDMA, GSM, UMTS, or other cellular communication network). In some embodiments, the network interface may enable a point-to-point connection with another device, for example, via a Universal Serial Bus (USB), 1394 FireWire, serial or parallel connection, or similar interface. Some embodiments of a suitable computing device may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include a data store in addition to or in place of a network interface.

[0154] Some embodiments of a suitable computing device may include or be in communication with a number of external or internal devices, such as a mouse, a CD-ROM, a DVD, a keyboard, a display, an audio speaker, one or more microphones, or any other input or output device. For example, the computing device may be in communication with a variety of user interface devices and displays. The displays may use any suitable technology, including, but not limited to, LCD, LED, CRT, etc.

[0155] The set of instructions for execution by the computer system may include various commands that instruct a processing machine to perform particular tasks (e.g., steps constituting the methods of the present technology). The set of instructions may be in the form of a software program. Moreover, the software may be in the form of a collection of separate programs, a program module with a larger program, or a portion of a program module, as in the present technology. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be in response to user commands, results of previous processing, or a request made by another processing machine.

[0156] While the present invention has been disclosed with reference to certain embodiments, many modifications, variations and alterations to the described embodiments are possible without departing from the scope and spirit of the invention, as defined in the appended claims. Thus, the present invention is not intended to be limited to the above-described embodiments, but has full scope defined by the language of the following claims and equivalents thereof. EXAMPLES

[0157] The results presented in the Examples below demonstrate detection of few cells, as few as one Listeria bacterium, with reduced time to results.

[0158] Example 1. Generation and isolation of indicator phages from Listeria-specific bacteriophages Indicator phages Listeria-specific LMA4.NANOLUC, LMA8.NANOLUC, and other Listeria bacteriophages were generated through homologous recombination using procedures as previously described. See Figures 1-3, which show and describe recombinant Listeria phages derived from LMA4 and LMA8.

[0159] The genome sequences of these phages were obtained through whole genome sequencing using the Illumina MiSeq system with de novo sequence assembly. Based on previously known and annotated genomes of related phages, the late gene regions and major capsid protein genes were located on the new phage genomes. Plasmids were designed and synthesized to insert NANOLUC® with appropriate late gene promoters and ribosome binding sites flanked by approximately 200-500 bp of matching phage sequences to facilitate homologous recombination.

[0160] Target bacteria were transformed with the homologous recombination plasmid under appropriate antibiotic selection and infected with their respective wild-type phages to allow homologous recombination with the plasmid. After homologous recombination to generate the recombinant bacteriophage genome, a series of titration and enrichment steps were used to isolate specific recombinant bacteriophages expressing NANOLUC® as previously described.

[0161] Finally, large-scale production was performed to obtain high-titer stocks suitable for use in Listeria spp. detection assays. Cesium chloride isopycnic density gradient centrifugation was used to separate phage particles from contaminating luciferase protein and reduce background. In another embodiment, sucrose isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein and reduce background.

[0162] Example 2. Inoculated Sponge Samples - Listeria Sponge Assay EZ Reach polyurethane sponge samples were pre-wetted with Dey / Engley broth and spiked with <1 CFU of Listeria monocytogenes or 100 CFU of challenge bacteria (Cronobacter sakazakii) diluted from overnight cultures.

[0163] The sponge handle was broken off and the sponge was placed into the medium in the bag. Buffered Listeria Enrichment Broth (BLEB) (Remel) medium (10 mL or 90 mL) was added to cover as much of the sponge as possible. The sponge was then gently massaged to release the bacteria into the medium and enrichment was continued for 16-18 or 24 hours at 35°C. After enrichment, the sponge was gently massaged / squeezed to remove the liquid and then removed from the medium in the bag. The bag was then gently massaged to mix the contents. A 150 μL aliquot was transferred to a 96-well plate. The sponge was then placed into medium at 35°C for further enrichment if necessary.

[0164] Sponge samples were tested with Listeria phage cocktail after 1 and 4 hours of infection. Briefly, phage reagent (10 μL) was added to the samples and the samples were incubated at 30° C. for 1 or 4 hours. Finally, 65 μL of luciferase master mix reagent was added to each well and mixed gently by pipetting up and down. Samples were read on a luminometer (GloMax96) instrument for 3 minutes after substrate addition (i.e., luminescence was detected). Sponges / swabs were placed back into the bag / tube and enrichment was continued for a total of 24 hours at 35° C. Aliquots could be taken, further enriched, and tested again if necessary.

[0165] The results are shown in Figures 5A and 5B. A signal to background ratio (S / B) greater than 3 was considered positive. The background level was determined to be 100 RLU based on previous phage characterization. These experiments show that the Listeria phage assay can detect 1 CFU spike of L. monocytogenes ATCC 19115 after 16-18 hours of enrichment and 1 hour of infection. Figure 5A (10 mL medium) shows that sponge samples 1 and 4 were negative for detection of Listeria (i.e., S / B<3.0). Sponge samples 2, 3, 5, and the 10 CFU control were all positive for all enrichment and infection times. Figure 5B (90 mL medium) shows that sponge 1 was negative for detection of Listeria, while all other samples had positive detection. These data show that the sponge with 10 mL spiked medium produced a better signal with a higher relative S / B than the sample with 90 mL spiked medium.

[0166] Thus, the experiments show that it is possible to detect a 1 CFU addition from an overnight culture in all conditions tried (i.e., 16-18 h enrichment-1 h infection, 16-18 h enrichment-4 h infection, 24 h enrichment-1 h infection, and 24 h enrichment-4 h infection).

[0167] Example 3. Environmental Surface Samples - Listeria Sponge Assay Stainless steel surfaces were incubated with the indicated number of cells in culture medium. Cells were allowed to dry on the surface and maintained at room temperature for 18-24 hours, after which they were wiped with an EZ Reach polyurethane sponge sample pre-wetted with Letheen medium (World BioProducts). Listeria monocytogenes 19115 was used as the target and Staphylococcus aureus 12600 was used as the challenge strain.

[0168] The sponge handle was broken off and the sponge was placed back into the medium in the bag. Buffered Listeria Enrichment Broth (BLEB) (Remel) medium (20 mL) was added to cover as much of the sponge as possible. The sponge was then gently massaged to release the bacteria into the medium and enrichment was continued for 20 hours at 35°C. After enrichment, the sponge was gently massaged and squeezed to remove liquid and then removed from the medium in the bag. The bag was gently massaged to mix the contents. 150 μL aliquots were transferred to 96-well plates. The sponge was placed back into medium if further enrichment was required and incubated at 35°C.

[0169] Sponge samples were tested with Listeria phage cocktail after 1 and 4 hours of infection. Briefly, phage reagent (10 μL) was added to the samples and incubated at 30° C. for 4 hours. Finally, 65 μL of luciferase master mix reagent was added to each well and mixed gently by pipetting up and down. Samples were read on the GloMax96 instrument for 3 minutes after substrate addition (i.e., luminescence was detected).

[0170] Phage reagent (10 μL) was added to the samples and incubated for 4 hours at 30° C. Finally, 65 μL of luciferase master mix reagent was added to each well and mixed gently by pipetting up and down. Samples were read (i.e., luminescence was detected) on a GloMax96 instrument for 3 minutes (180 seconds) after substrate addition.

[0171] FIG. 6 shows the RLUs generated from stainless steel surface swabs. Samples generating RLUs >300 were considered positive. These data show that the Listeria phage assay can detect a 100 CFU surface inoculum of L. monocytogenes at 20 hours enrichment and 4 hours infection in the presence of non-Listeria bacteria present at CFU levels 10-fold greater than the target Listeria bacteria. Sample 2 and the negative control were negative for detection of L. monocytogenes. All other samples (including the positive control) were positive. Compared to the spiked sponge experiment, the stainless steel swabs required a longer enrichment period and higher CFU levels for positive detection. This may be due to several variables, including increased injury to surface-inoculated cells when compared to those derived from overnight cultures. Surface-inoculated cells typically have a significant loss of viability. Also, recovery of cells forming the surface on the sponge may be highly variable.

Claims

1. A composition comprising at least one recombinant bacteriophage, said recombinant bacteriophage being constructed from LMA4, said bacteriophage comprising: (i) an indicator gene inserted into the late gene region of the bacteriophage genome; (ii) an exogenous promoter controlling transcription of the indicator gene, the exogenous promoter being a bacteriophage late gene promoter; Including, the indicator gene is not linked to a gene encoding a structural bacteriophage protein and does not result in a fusion protein; expression of the indicator gene results in an indicator protein product; The composition, wherein said recombinant bacteriophage specifically infects Listeria spp.

2. 10. The composition of claim 1, wherein the composition further comprises at least one recombinant bacteriophage constructed from one of LMA8, A511, and P70 bacteriophages.

3. The composition of claim 1 , wherein the indicator gene encodes a soluble protein product that produces an endogenous signal or a soluble enzyme that produces a signal upon reaction with a substrate.

4. 2. The composition of claim 1, wherein the recombinant bacteriophage comprises a ribosome entry site, and the bacteriophage late gene promoter and the ribosome entry site are located in an untranslated region upstream of the indicator gene.

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