Methods and systems for rapid detection of microorganisms using recombinant infectious agents expressing indicator subunits

Abstract

Disclosed herein are methods and systems for rapidly detecting microorganisms, such as bacteria, in a sample. Also disclosed are genetically modified bacteriophages that contain an indicator gene encoding a subunit of an indicator protein. The specificity of the bacteriophage allows for detection of specific target bacteria and amplifies the indicator signal to optimize assay sensitivity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 898,945, filed September 11, 2019. The disclosures of U.S. Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, and 15 / 409,258 are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure relates to compositions, methods, systems, and kits for detecting microorganisms using infectious agents. Background Art

[0004] There is a strong interest in improving the speed and sensitivity of the bacteria, viruses and other microorganisms in detecting organisms, food, water and clinical samples. Microbial pathogens can cause serious morbidity in humans and livestock, and huge economic losses. In addition, in view of causing the outbreak of life-threatening or fatal diseases by taking in food contaminated by certain microorganisms, microbial detection is a high priority of the Food and Drug Administration (FDA) and Centers for Disease Control (CDC) and the United States Department of Agriculture (USDA), such as Escherichia coli, Cronobacter species, Salmonella species, Listeria species or Staphylococcus species.

[0005] Traditional microbiological tests for detecting bacteria rely on non-selective and selective enrichment cultures, which are then plated on selective media and further tested to confirm suspicious colonies. Such procedures may take several days. A variety of rapid methods have been studied and introduced into practice to reduce time requirements. However, these methods have shortcomings. For example, the technology related to direct immunoassays or gene probes generally requires an overnight enrichment step to obtain sufficient sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step, and therefore can have very high sensitivity and selectivity; however, the sample size that can be economically tested with PCR is limited. Using diluted bacterial suspensions, most small subsamples will not contain cells, and therefore still require purification and / or lengthy enrichment steps.

[0006] The time required for traditional bioenrichment depends on the growth rate of the target bacterial population in the sample, the effects of the sample matrix, and the desired sensitivity. In practice, most high-sensitivity methods use overnight incubation and require approximately 24 hours in total. Due to the time required for incubation, these methods can take up to three days, depending on the organism to be identified and the source of the sample. This lag time is often unsuitable because contaminated food, water, or other products may have entered livestock or humans. In addition, the increase in antibiotic-resistant bacteria and biodefense concerns make the rapid identification of bacterial pathogens in water, food, and clinical samples a key global priority.

[0007] Therefore, 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

[0008] Embodiments of the present disclosure include compositions, methods, systems, and kits for detecting microorganisms. For example, the present disclosure can be embodied in various ways.

[0009] In some aspects, the present disclosure includes recombinant indicator phages comprising an indicator gene inserted into a phage genome, wherein the indicator gene encodes a peptide subunit of an indicator protein (indicator protein product). In certain embodiments, the indicator phage comprises a genetically modified phage genome derived from a phage that specifically recognizes a specific target bacterium.

[0010] In some embodiments of the recombinant indicator phage, the peptide subunit (marker subunit) is part of a split reporter enzyme system, wherein the reporter enzyme (i.e., indicator protein) is a luciferase. The luciferase can be naturally occurring, such as Oplophorus luciferase, firefly luciferase, Lucia luciferase, or Renilla luciferase, or it can be a genetically engineered luciferase such as

[0011] Also disclosed herein are methods for preparing indicator phage. Some embodiments include selecting a wild-type phage that specifically infects a target pathogen; preparing a homologous recombinant plasmid / vector containing an indicator gene; transforming the homologous recombinant plasmid / vector into the target pathogen; infecting the transformed target pathogen with the selected wild-type phage, thereby causing homologous recombination between the plasmid / vector and the phage genome; and isolating a specific clone of the recombinant phage.

[0012] In another aspect, the present disclosure includes a method for detecting a specific target bacterium in a sample, comprising the steps of: incubating the sample with a recombinant indicator phage comprising an indicator gene, wherein the indicator gene encodes a first subunit of an indicator protein, thereby producing a certain amount of progeny phage and expressing the first subunit; lysing the bacteria in the sample to release a certain amount of progeny phage and the first subunit; incubating the lysed sample in the presence of a detection reagent, wherein the detection reagent comprises a second subunit of the indicator protein, thereby allowing the first subunit and the second subunit to reconstitute to form an indicator protein complex; and detecting the indicator protein complex, wherein a positive detection of the indicator protein complex indicates the presence of the specific target bacterium in the sample.

[0013] In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions conducive for 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 obtain a result is less than 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours. In some embodiments, the ratio of the signal produced by the detection indicator to the background 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 specific bacteria in a food safety industry standard size sample.

[0014] Additional embodiments include systems and kits for detecting specific target bacteria, wherein the system or kit includes an indicator phage derived from a phage specific for the specific target bacteria. These systems or kits can include features described for the phage, compositions, and methods of the present disclosure. In other embodiments, the present disclosure includes a non-transient computer-readable medium for use in accordance with the methods or systems of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present disclosure may be better understood by reference to the following non-limiting drawings.

[0016] Figure 1 Depicted are indicator phage constructs according to embodiments of the present disclosure illustrating the insertion of a genetic construct comprising a promoter, RBS, and an indicator gene (soluble HiBiT) into TSP12 phage downstream of the major capsid protein.

[0017] Figure 2 Depicted are indicator phage constructs according to embodiments of the present disclosure illustrating the insertion of a genetic construct comprising a linker, an HRV 3C site, and an indicator gene (HiBiT) into TSP12 phage downstream of the major capsid protein.

[0018] Figure 3 Depicted are indicator phage constructs according to embodiments of the present disclosure illustrating the insertion of a genetic construct comprising a linker, a PS protease site, and an indicator gene (HiBiT) into the N-terminus of the Soc protein in TSP12 phage.

[0019] Figure 4 Tables are provided detailing homologous recombination constructs prepared using parental phage TSP1, TSP12, SEA1, and T7Select according to embodiments of the present disclosure.

[0020] Figure 5 The example illustrates the optimization of TSPl.sHiBiT phage concentration for detection of Salmonella typhimurium by 2 hours incubation according to an embodiment of the present disclosure.

[0021] Figure 6 Optimization of TSPl.sHiBiT phage concentration for detection of Salmonella typhimurium by 3 hours incubation is illustrated according to embodiments of the present disclosure.

[0022] Figure 7 The example illustrates the optimization of TSP12.sHiBiT phage concentration for detection of Salmonella bongori by 2 hours incubation according to embodiments of the present disclosure.

[0023] Figure 8 Optimization of TSP12.sHiBiT phage concentration for detection of Salmonella bongori by 4-hour incubation is illustrated according to embodiments of the present disclosure.

[0024] Figure 9The example illustrates the optimization of TSP12.HiBiT-PS-Soc phage concentration for detection of Salmonella bongori by 2 hours incubation according to embodiments of the present disclosure.

[0025] Figure 10 The example illustrates the optimization of TSP12.HiBiT-PS-Soc phage concentration for detection of Salmonella bongori by 2 hours incubation according to embodiments of the present disclosure. Detailed Description of the Invention

[0027] Disclosed herein are compositions, methods and systems that demonstrate surprising sensitivity to target microorganisms (e.g., E. coli, Cronobacter species, Salmonella species, Listeria species, or Staphylococcus species) in detection test samples (e.g., organisms, food, water, and the environment). Detection can be achieved within a shorter timeframe than previously thought in assays without enrichment cultures using genetically modified infectious agents or in some embodiments of the minimum incubation time for potential propagation of microorganisms. Equally surprising is the successful use of potential high multiplicity of infection (MOI) or high concentrations of plaque forming units (PFU) to incubate the test sample. Previously, it was believed that such high phage concentrations (PFU / mL) were harmful in bacterial detection assays because they were said to result in "self-lysis." However, high concentrations of phage contribute to discovery, binding, and infection of a small amount of target cells.

[0028] The compositions, methods, systems and kits of the present disclosure may include for detecting infectious agents of microorganisms such as Escherichia coli, Cronobacter species, Salmonella species, Listeria species or Staphylococcus species. A recombinant indicator phage comprising an indicator gene inserted into the phage genome, wherein the indicator gene encodes a peptide subunit of an indicator protein. In certain embodiments, the expression of the indicator gene during phage replication after infection of the host bacteria results in the production of a soluble peptide subunit of the indicator protein. In alternative embodiments, it is a fusion peptide subunit of an indicator protein. In certain embodiments, the indicator gene can be inserted into the late gene (i.e., Class III) region of the phage.

[0029] In some embodiments, the present disclosure includes a method for detecting a specific target bacterium in a sample, comprising the steps of: incubating the sample with a recombinant indicator phage comprising an indicator gene, wherein the indicator gene encodes a first subunit of an indicator protein, thereby producing a certain amount of progeny phage expressing the first subunit; lysing a certain amount of progeny phage; incubating the lysed progeny phage in the presence of a detection reagent, wherein the detection reagent comprises a second subunit of the indicator protein, thereby allowing the first subunit and the second subunit to reconstitute to form an indicator protein complex; and detecting the indicator protein complex, wherein a positive detection of the indicator protein complex indicates the presence of the specific target bacterium in the sample.

[0030] In certain embodiments, the present disclosure may include a system. The system may include at least some of the compositions of the present disclosure. In addition, the system may include at least some components for performing the methods. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present disclosure may include a system for rapidly detecting specific target bacteria in a sample, comprising: components for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent includes an indicator moiety; and components for detecting the indicator moiety. In yet other embodiments, the present disclosure includes software for the method or system.

[0031] Therefore, some embodiments of the present disclosure address the need by using phage-based methods for amplifying detectable signals indicating the presence of bacteria. In certain embodiments, as few as a single bacterium is detected. The principles employed herein can be applied to the detection of a variety of microorganisms. Due to the many binding sites for infectious agents on the surface of microorganisms, the ability to produce one hundred or more progeny of infectious agents during the infection process, and the potential for high-level expression of the encoded indicator portion, infectious agents or indicator portions can be more easily detected than the microorganisms themselves. In this way, embodiments of the present disclosure can achieve huge signal amplification even from a single infected cell.

[0032] Some aspects of the present disclosure utilize the high specificity of binding agents that can bind to specific microorganisms, such as binding components of infectious agents, as a tool for detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the present disclosure utilizes the high specificity of infectious agents such as bacteriophages.

[0033] In some embodiments, detection is achieved by an indicator portion associated with a binding agent specific for the target microorganism. For example, the infectious agent can include a gene encoding an indicator protein or its subunits. In some embodiments, the indicator can be encoded by an infectious agent (e.g., a bacteriophage), and the bacteriophage is designated as an indicator phage.

[0034] Some embodiments of the present disclosure disclosed and described herein utilize the discovery that a single microorganism can be combined with a specific recognition agent (e.g., phage). After phage infection and replication, progeny phage can be detected by an indicator gene expressed during phage replication. This principle allows the specific recognition based on microbial surface receptors to amplify the indicator signal from one or several cells. For example, by exposing a single cell of bacteria to multiple phages, then allowing phage amplification and encoded indicator gene products or high-level expression of the subunits of the indicator protein during replication, the indicator signal is amplified so that a single bacterium is detectable.

[0035] The embodiments of the methods and systems of the present disclosure can be applied to detect and quantify a variety of microorganisms (e.g., bacteria) in a variety of situations, including but not limited to detecting pathogens from food, water, and commercial samples. The methods of the present disclosure provide rapid high detection sensitivity and specificity. In some embodiments, detection can be performed within a single replication cycle of a bacteriophage, which is unexpected.

[0036] definition

[0037] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. Generally speaking, the nomenclature and technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well known in the art and commonly used. Unless otherwise indicated, known method and technology are usually carried out according to conventional methods well known in the art, and as described in the various general and more specific references discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as usually completed in this area or as described herein. The nomenclature used in conjunction with laboratory procedures as described herein and technology is well known in the art and commonly used.

[0038] The following terms, unless otherwise specified, shall be understood to have the following meanings:

[0039] As used herein, the terms "a," "an," and "the" may refer to one or more unless specifically stated otherwise.

[0040] Use of the term "or" is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0041] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the equipment, the method being employed to determine the value, or variation that exists between samples.

[0042] As used herein, "solid support" or "support" refers to a structure that provides a substrate and / or surface to which biomolecules can be bound. For example, a solid support can be an assay well (i.e., a microtiter plate or multiwell plate), or a solid support can be a location on a filter, array, or mobile support such as beads or membranes (e.g., filter plates, latex particles, paramagnetic particles, or lateral flow strips).

[0043] As used herein, a "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) target molecule. The interaction can be non-covalent, for example, due to hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it 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.

[0044] As used herein, the term "bioluminescence" refers to the generation and emission of light by a chemical reaction catalyzed or facilitated by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescent complex). In typical embodiments, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted to an unstable form by the bioluminescent entity; the substrate subsequently emits light.

[0045] As used herein, the term "complementary" refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that are capable of hybridizing, dimerizing, or otherwise forming a complex with each other. For example, a "complementary peptide and polypeptide" are capable of forming a complex together. Complementary elements may require assistance to form a complex (e.g., from an interacting element), e.g., placing the elements in an appropriate conformation for complementation, co-localizing complementary elements, reducing the complementary interaction energy, etc.

[0046] As used herein, the term "complex" refers to an aggregate or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. On the one hand, "contact", or more specifically, "direct contact" refers to two or more molecules that are close enough to allow attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, etc., to dominate the interaction of molecules. In such an aspect, a complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions so that the complex is thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed state of its constituent molecules). As used herein, unless otherwise indicated, the term "complex" refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof).

[0047] As used herein, the term "non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, if an entity does not exhibit detectable luminescence in a given assay, it can be referred to as non-luminescent. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent." For example, a non-luminescent polypeptide is substantially non-luminescent, exhibiting a luminescence reduction of, for example, 10 times or more (e.g., 100 times, 200 times, 500 times, 1×10Sup3 / Sup-times, 1×10Sup4 / Sup-times, 1×10Sup5 / Sup-times, 1×10Sup6 / Sup-times, 1×10Sup7 / Sup-times, etc.) compared to a complex of an NLpoly with its non-luminescent complement peptide. In some embodiments, an entity is "non-luminescent" if any light emission is small enough to not produce an interfering background for a particular assay.

[0048] As used herein, the terms "non-luminescent peptides" and "non-luminescent polypeptides" refer to peptides and polypeptides that exhibit essentially no luminescence (e.g., in the presence of a substrate) or an amount of luminescence that is below noise, or 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10Sup3 / Sup-fold, 1×10Sup4 / Sup-fold, 1×10Sup5 / Sup-fold, 1×10Sup6 / Sup-fold, 1×10Sup7 / Sup-fold, etc.) compared to a significant signal (e.g., luminescent complex) under standard conditions (e.g., physiological conditions, assay conditions, etc.) and using typical instruments (e.g., luminometers, etc.). In some embodiments, such non-luminescent peptides and polypeptides are assembled according to the standards described herein to form a bioluminescent complex. As used herein, a "non-luminescent element" is a non-luminescent peptide or non-luminescent polypeptide. The term "bioluminescent complex" refers to an assembled complex of two or more non-luminescent peptides and / or non-luminescent polypeptides. The bioluminescent complex catalyzes or is capable of converting the substrate of the bioluminescent complex into an unstable form; the substrate subsequently emits light. When uncomplexed, the two non-luminescent elements that form the bioluminescent complex can be referred to as a "non-luminescent pair." If the bioluminescent complex is formed from three or more non-luminescent peptides and / or non-luminescent polypeptides, the uncomplexed components of the bioluminescent complex can be referred to as a "non-luminescent group."

[0049] As used herein, "analyte" refers to a molecule, compound, or cell being measured. In certain embodiments, the target analyte may interact with a binding agent. As described herein, the term "analyte" may refer to a target protein or peptide. The analyte may be an agonist, antagonist, or modulator. Alternatively, the analyte may not have a biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptide mimetics, organic compounds, and the like.

[0050] As used herein, a "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicator protein" or "indicator protein product" or "indicator protein complex" or "indicator moiety" refers to a molecule that can be measured in a quantitative assay. For example, the indicator protein can comprise an enzyme that can be used to convert a substrate into a product that can be measured. The indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that produces bioluminescent emission. Alternatively, the indicator moiety can be a radioactive isotope that can be quantified. Alternatively, the indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0051] As used herein, "bacteriophage" or "phage" includes one or more of a variety of bacterial viruses. In this disclosure, the terms "bacteriophage" and "phage" include viruses such as mycobacteriophages (e.g., for TB and paraTB), fungal phages (e.g., for fungi), mycoplasma phages, and any other term referring to viruses that can invade living bacteria, fungi, mycoplasmas, protozoa, yeasts, and other microscopic living organisms and use them to replicate themselves. Here, "microscopic" means a maximum size of one millimeter or less. Phages are viruses that have evolved in nature to use bacteria as a means of self-replication. Phages achieve this by attaching themselves to bacteria and injecting their DNA (or RNA) into the bacteria and inducing it to replicate the phage hundreds or even thousands of times. This is called phage amplification.

[0052] As used herein, " late gene region " refers to the region in the viral genome that is transcribed in the late stage of the viral life cycle. The late gene region generally includes the most abundantly expressed genes (for example, structural proteins assembled into phage particles). Late genes are synonymous with class III genes, including genes with structure and assembly functions. For example, late genes (synonymous with class III) are transcribed in phage T7, for example, from 8 minutes after infection until lysis, class I (for example RNA polymerase) starts as early as from 4-8 minutes, and class II starts from 6-15 minutes, so the time of II and III overlaps. Late promoters are naturally located and active promoters in this late gene region.

[0053] As used herein, "enrichment culture" refers to traditional culture, such as incubation in a medium that is conducive to the growth of microorganisms, and should not be confused with other possible uses of the term "enrichment," such as removing liquid components of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not involve traditional promotion of microbial growth. In some embodiments of the methods described herein, culturing for a period of time can be used to perform enrichment.

[0054] As used herein, "recombinant" refers to genetic (ie, nucleic acid) modifications, usually performed in a laboratory, to bring together genetic material that would not otherwise be found. The term is used interchangeably with the term "modified" herein.

[0055] As used herein, "RLU" refers to the number of units measured by a photometer (e.g., 96) or similar instruments that detect light. For example, luciferase is combined with an appropriate substrate (e.g. and ) is typically reported in RLU detected.

[0056] As used herein, "time to result" refers to the total time from the start of sample incubation to the generation of a result. Time to result does not include any confirmatory testing time. Data collection can begin immediately after the result is generated.

[0057] As used herein, "reporter gene" or "indicator gene" can refer to a complete gene or a portion of a gene. For example, the indicator gene or reporter gene used herein can include a nucleotide sequence encoding a smaller peptide subunit, that is, it is transcribed and translated into a partial protein.

[0058] sample

[0059] Each embodiment of the methods and systems of the present disclosure can allow for rapid detection and quantification of microorganisms in a sample. For example, the methods according to the present disclosure can be performed in a shortened period of time and with excellent results.

[0060] Microorganisms detected by the methods and systems of the present invention include pathogens of natural, commercial, medical, or veterinary concern. Such pathogens include Gram-negative bacteria, Gram-positive bacteria, and mycoplasmas. Any microorganism for which an infectious agent specific for a particular microorganism has been identified can be detected by the methods of the present invention. Those skilled in the art will appreciate that, other than the availability of the necessary specific infectious agent / microorganism pair, there is no limitation to the application of this method.

[0061] Bacterial cells detectable by the present disclosure include, but are not limited to, bacterial cells that are food or waterborne pathogens. Bacterial cells detectable by the present disclosure include, but are not limited to, all species of Salmonella, all strains of Escherichia coli, Cronobacter, Staphylococcus, all species of Listeria, including but not limited to all species of Listeria monocytogenes (L.monocytogenes) and Campylobacter. Bacterial cells detectable by the present disclosure include, but are not limited to, bacterial cells that are pathogens of medical or veterinary significance. Such pathogens include, but are not limited to, Bacillus spp., Bordetella pertussis, Campyobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Shigella sonnei, Staphylococcus aureus, and Streptococcus spp. In some embodiments, the bacterial cells detectable by the present invention include antibiotic-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)).

[0062] The sample can be an environmental sample or a food sample or a water sample. Some embodiments can include medical or veterinary samples. The sample can be a liquid, solid or semi-solid. The sample can be a swab on a solid surface. The sample can include environmental materials, such as a water sample, or a filter from an air sample or an aerosol sample from a cyclone collector. The sample can be vegetables, meat, fish, poultry, peanut butter, processed food, infant formula, milk powder, tea, starch, eggs, milk, cheese or other dairy products. Medical or veterinary samples include but are not limited to blood, sputum, cerebrospinal fluid and fecal samples and different types of swabs.

[0063] In some embodiments, the sample can be used directly in the detection method of the present disclosure without preparation, concentration or dilution. For example, liquid samples, including but not limited to milk and juice, can be directly assayed. The sample can be diluted or suspended in a solution, which can include but is not limited to a buffer solution or a bacterial culture medium. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing or immersing the solid in the liquid. The sample should be maintained in a pH range that promotes phage attachment to host bacterial cells. The sample should also contain appropriate concentrations of divalent and monovalent cations, including but not limited to Na + Mg 2+ and Ca 2+ Preferably, the sample is maintained at a temperature that supports the viability of any pathogen cells contained in the sample.

[0064] Preferably, throughout the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. During the step in which phage attaches to bacterial cells, it is preferred that the sample be maintained at a temperature that is conducive to phage attachment. During the step in which phage replicates within infected bacterial cells or lyses such infected cells, it is preferred that the sample be maintained at a temperature that promotes phage replication and host lysis. Such a temperature is at least about 25 degrees Celsius (C), more preferably no greater than about 45 degrees Celsius, and most preferably about 37 degrees Celsius.

[0065] The assay can include various suitable control samples. For example, a control sample without phage or a control sample without bacteria but containing phage can be assayed as a control for background signal levels.

[0066] indicator phage

[0067] As described in more detail herein, the compositions, methods, systems, and kits of the present invention may include infectious agents for detecting pathogenic microorganisms. In certain embodiments, the present disclosure includes recombinant indicator phages, wherein the phage genome is genetically modified to include an indicator gene or reporter gene. In some embodiments, the present invention may include a composition comprising a recombinant phage having an indicator gene incorporated into the phage genome, wherein the indicator gene encodes a peptide subunit of an indicator protein.

[0068] The recombinant indicator phage may include a reporter gene or an indicator gene or an indicator peptide subunit. In some embodiments of the indicator phage, the indicator or peptide subunit gene encodes a fusion protein. For example, the indicator or indicator peptide subunit can be fused with a phage capsid protein so that the indicator is expressed as part of the phage capsid. The indicator can also be fused with another protein to produce a soluble molecule. In other embodiments of the indicator phage, the indicator gene does not encode a fusion protein. For example, in certain embodiments, the expression of the indicator gene during phage replication after infection of the host bacteria results in a non-fused soluble indicator protein product. In certain embodiments, the indicator or indicator peptide or polypeptide subunit gene can be inserted into the late gene region of the phage. Late genes are typically expressed at a higher level than other phage genes because they encode structural proteins. The late gene region can be a class III gene region and can include genes for major capsid proteins.

[0069] Some embodiments include designing (and optionally preparing) sequences for homologous recombination in the downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) sequences for homologous recombination in the upstream of the major capsid protein gene. In some embodiments, the sequence comprises a codon-optimized reporter gene preceded by a non-translated region. The non-translated region may comprise a phage late gene promoter and a ribosome entry site.

[0070] In some embodiments, the indicator phage is derived from selected wild-type phages: Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage ε15, Salmonella phage SEA1, Salmonella phage TSPl, Salmonella phage TSP12, Salmonella phage Spn1s, Salmonella phage P22, Listeria phage LipZ5, Listeria phage P40, Listeria phage vB_LmoM_ AG20, Listeria phage P70, Listeria phage A511, Listeria phage P100, Listeria phage LMA8, Listeria phage LMA4, Staphylococcus phage P4W, Staphylococcus phage K, Staphylococcus phage Twort, Staphylococcus phage SA97, Escherichia coli O157:H7 phage CBA120, or another phage having a similar affinity to the selected wild-type phage Salmonella phage S PN1S, Salmonella phage 10, Salmonella phage ε15, Salmonella phage SEA1, Salmonella phage TSP1, Salmonella phage TSP12, Salmonella phage Spn1s, Salmonella phage P22, Listeria phage LipZ5, Listeria phage P40, Listeria phage vB_LmoM_AG20, Listeria phage P70, Listeria phage A511, Staphylococcus phage In some embodiments, the indicator phage is derived from a phage that is highly specific for a particular pathogenic microorganism. Genetic modification can avoid the deletion of wild-type genes, so the modified phage can be more similar to the wild-type infectious agent than many commercially available phages. Environmentally derived phages may be more specific for bacteria found in the environment and therefore genetically different from commercially available phages.

[0071] In some embodiments, the recombinant phage comprises a binding domain that is ≥95% homologous to the binding domain of any of the following phages: Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage ε15, Salmonella phage SEA1, Salmonella phage TSPl, Salmonella phage TSP12, Salmonella phage Spn1s, Salmonella phage P22, Listeria phage LipZ5, Listeria phage P40, Listeria phage vB_LmoM_AG20, Listeria phage P70, Listeria phage A511, Staphylococcus phage P4W, Staphylococcus phage K, Staphylococcus phage Twort, Staphylococcus phage SA97, or Escherichia coli O157:H7 phage CBA120.

[0072] In some cases, the indicator phage is derived from a phage that is highly specific to a particular pathogenic microorganism. In some embodiments, the indicator phage is derived from T7Select. T7Select is a commercially available phage display system from Novagen. T7 is a well-characterized prototype phage that infects Escherichia (Escherichia), from the Podoviridae (Podoviridae). The capsid of T7 phage is composed of a 9:1 ratio of 2 isotypes (gp10a and gp10b) of MCP. Gp10a and gp10b are caused by frameshifting in translation, and this frameshifting can be modulated to produce gp10a:gp10b isomers of different ratios. It is a cloning plasmid containing the entire T7 genome. Peptides or proteins of a certain length can be cloned into the C-terminus of gp10b and expressed at high (415), medium (5-10), or low (up to 1) copy numbers per phage.

[0073] In some embodiments, the indicator phage is derived from TSP12, a Salmonella phage specific for Salmonella bongori strains. This phage is most closely related to the Enterobacter RB51 phage belonging to the genus Tequatrovirus. This genus includes the well-characterized and studied Escherichia coli T4 virus. This phage has numerous capsid structures, with detailed descriptions of protein components and conformations. TSP12 has several structural candidates for tagging using peptide or polypeptide marker subunits, including but not limited to the major capsid protein (gp23) and the accessory small outer capsid protein (gpSoc).

[0074] In some embodiments, the indicator phage is derived from Salmonella phage SEA1. SEA1 is most closely related to Salmonella phage vB_SenM-S16 [GenBank: HQ331142.1] of the genus Gelderlandvirus. SEA1 has several structural candidate genes for tagging with peptide or polypeptide marker subunits, including but not limited to the major capsid protein, the head vertex protein, the head outer capsid protein, and the minor outer capsid protein. The major capsid protein can be tagged at either the amino (N) or carboxyl (C) terminus.

[0075] In some embodiments, the indicator phage is derived from the Salmonella phage TSP1. TSP1 is most closely related to phages of the Salmonella and Escherichia Kuttervirus genera. TSP1 phage has at least one candidate gene for tagging with a peptide or polypeptide marker subunit, including but not limited to a single major prohead protein.

[0076] Genetic modification can avoid the loss of wild-type genes, so that the modified phage can remain more similar to the wild-type infectious agent than many commercially available phage. Environmentally derived phages may be more specific to bacteria found in the environment and therefore be genetically different from commercially available phages.

[0077] In addition, phage genes that are considered to be non-essential can have unrecognized functions. For example, genes that are obviously non-essential can have important functions in improving the lysis amount, such as subtle cutting, fitting or trimming functions in assembly. Therefore, deleting genes to insert indicators may be harmful. Most phages can package up to 10% more DNA than their natural genomes. Different viruses, including phages, have different lysis amounts. The lysis amount depends to a great extent on the host, multiplicity of infection (MOI) and growth conditions. The lysis amount of lambda phage and other phages (such as T4, T5 and T7) is about 100-300. In some embodiments, the selected phage has a lysis amount of at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 250, 375, 400, 425, 450, 475 or 500 PFU / cell. For example, T7 has a lysis capacity of 180 PFU / cell; T4 has a lysis capacity of 130 PFU / cell; and CBA120 has a lysis capacity of 440 PFU / cell. A smaller lysis capacity means fewer progeny phages can be used to produce the indicator protein product. Therefore, using phages with a larger lysis capacity is beneficial for amplifying the signal and improving the sensitivity of the assay.

[0078] Small phages package smaller genomes and are therefore less tolerant of additional transgenes. Another possible advantage of small reporter genes is that they can be expressed in higher quantities because each copy of the protein requires fewer of the finite cellular resources. However, the HiBiT tag alone may be too small to be expressed or folded correctly.

[0079] Given these considerations, smaller indicator genes may be a more suitable choice for modifying phages, especially those with smaller genomes. The protein is only about 20 kDa (about 500-600 bp for encoding), while FLuc is about 62 kDa (about 1,700 bp for encoding). In contrast, the genome of T7 is about 40 kbp, while the genome of T4 is about 170 kbp. In some embodiments, the indicator gene encodes a reporter protein (e.g., In some embodiments, the use of smaller indicator genes (e.g., subunits of indicator proteins) allows multiple copies of the indicator gene to be inserted into the phage genome, thereby further amplifying the signal.

[0080] Protein complementation assays (PCA) provide a method for detecting the interaction of two biomolecules (e.g., polypeptide subunits). PCA can utilize two subunits of the same protein, such as an enzyme, which can be reconstituted into a functional, active protein when brought into proximity with each other. PCA involves the use of at least two subunits of a protein to detect a target protein. Thus, in some embodiments of the recombinant indicator phage, the indicator gene encodes a subunit of a split reporter protein. In some cases, the split reporter protein is a functional enzyme (e.g., luciferase or β-galactosidase). In further embodiments, the luciferase is In some embodiments, the split reporter (indicator protein) comprises a first polypeptide subunit (marker subunit) and a second polypeptide subunit (detection subunit). In a further embodiment, the marker subunit is complementary to the detection subunit. In some cases, the marker subunit is capable of binding to the detection subunit to form an indicator protein complex. Therefore, in some embodiments of the recombinant indicator phage, the indicator gene is inserted into the phage genome, wherein the indicator gene encodes a polypeptide subunit (marker subunit) of the indicator protein.

[0081] In some embodiments of the indicator phage, the gene encoding the marker subunit is inserted into the phage genome. In a further embodiment, during phage replication after infecting the target bacteria, the indicator gene causes the production of the indicator protein product, thereby allowing protein-protein interaction with the second subunit (detection subunit). In some embodiments, the marker subunit encounters the detection subunit to form a functional enzyme (e.g., luciferase). In a further embodiment, the functional enzyme produces a signal. In some cases, the generation of the signal requires the functional enzyme to contact a substrate.

[0082] Figure 1 A schematic diagram depicting the genome structure of one embodiment of the recombinant bacteriophage indicator phage TSP12.s.HiBiT of the present disclosure is shown. Figure 1 In the embodiment shown, the indicator protein is encoded by a soluble HiBiT gene inserted into the late (class III) gene region, which is expressed late in the viral life cycle. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. Therefore, in Figure 1 In the embodiment of the recombinant phage shown, the indicator gene (i.e., soluble HiBiT) is inserted into the late gene region, just after the major capsid protein (MCP) gene, and is a construct containing the HiBiT luciferase gene. Figure 1 As shown, the construct can include a late promoter to drive transcription and expression of the HiBiT gene. The construct can also include a composite untranslated region synthesized from several UTRs and stop codons in all three reading frames to ensure that HiBiT is not incorporated into the MCP gene product. This construct ensures the production of soluble HiBiT, so that expression is not limited by the amount of capsid protein inherent in phage display systems.

[0083] Figure 2 A schematic diagram depicting the genome structure of the recombinant phage indicator phage TSP12.MCP-PS-HiBiT of the present disclosure is shown. Figure 2 In the embodiment shown, the HiBiT gene is inserted into the C-terminus of MCP to produce an MCP-HiBiT fusion protein. MCP is located within the late (class III) gene region, which is expressed late in the viral life cycle. Late genes are typically expressed at higher levels than other phage genes because they encode structural proteins. Therefore, in Figure 2 In the embodiment of the recombinant phage shown, the indicator gene (i.e., HiBiT) is inserted into the late gene region, at the C-terminus of the MCP gene, and is a construct comprising the HiBiT luciferase gene. Figure 2As shown, the construct may comprise a linker and an HRV 3C protease cleavage site. The HRV 3C protease cleavage site allows for removal of the HiBit from the phage during phage production.

[0084] In some embodiments, The HiBiT Detection System (Promega Corporation) can be used to reconstitute luminescent enzymes to detect molecular proximity through binding interactions of enzyme components or subunits. The HiBiT assay system utilizes luciferase variants derived from Oplophorus In some embodiments, the indicator phage comprises an indicator gene, wherein the indicator gene is a HiBiT. When the HiBiT peptide encounters the LgBiT peptide, they reconstitute to form a full-length functional luciferase. In some embodiments, the detection reagent comprises a complementary polypeptide LgBiT, which spontaneously interacts with the HiBiT tag to reconstitute a brightly luminescent enzyme. In some embodiments, the indicator protein complex is combined with Promega's (an imidazopyrazinone substrate (furimazine)) combination can provide a robust signal with low background. In some embodiments, the detection reagent comprises

[0085] In some embodiments, the detection subunit has a high affinity for the labeling subunit. In further embodiments, the labeling subunit is capable of binding to the detection subunit to form an indicator complex. HiBiT binds tightly to LgBiT, thereby promoting the formation of the luciferase indicator complex. In some embodiments, the binding affinity (equilibrium dissociation constant (K)) between the labeling subunit and the detection subunit is D )) is at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 nM.

[0086] In some embodiments, each subunit exhibits little or no reporter activity. In certain embodiments, the labeling subunit and the detection subunit are non-luminescent or substantially non-luminescent. In other embodiments, the labeling subunit is luminescent. In addition, the reporter gene should not be endogenously expressed by the bacteria (i.e., not part of the bacterial genome), should produce a high signal-to-background ratio, and should be easy to detect in a timely manner.

[0087] In some embodiments, the gene encoding the marker subunit is inserted into the indicator phage genome. In certain embodiments, the marker subunit forms a fusion protein with a phage structural protein. These proteins are the most abundant proteins produced by phages because each phage particle contains tens or hundreds of copies of these molecules. In some embodiments, the marker subunit is fused to a phage capsid protein. In other embodiments, the marker subunit is fused to a phage tail fiber protein. In certain embodiments, the gene encoding the marker subunit is inserted into the late gene region of the phage. The late gene region can be a class III gene region and can include a gene for a major capsid protein. In some embodiments, the marker subunit forms a fusion protein with a capsid protein. For example, the marker subunit can be fused to a major capsid protein. The major capsid protein is present on the phage in multiple copies. For example, the T4 phage has approximately 1,000 copies of the major capsid protein, so the signal can be further amplified.

[0088] Reporter systems can be problematic because they can affect the proteins they interact with. In some embodiments, the reporter system has minimal steric burden on its fusion partner. In further embodiments, the reporter system has minimal impact on the affinity and association kinetics of the interacting target protein. In some embodiments, each subunit has been structurally optimized. In certain embodiments, the marker subunit is very small, thereby minimizing steric clashes on the fusion partner. In some cases, the detection subunit is optimized for stability. In some embodiments, it is advantageous for the marker subunit to be smaller than the detection subunit. Therefore, in certain embodiments, the marker subunit length is less than 50, 40, 30, 20, 15, 10 or 5 amino acids. In further embodiments, the detection subunit length is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 amino acids.

[0089] The genetic modification of the infectious agent can include the insertion, deletion or replacement of a small fragment of nucleic acid, a major part of a gene or an entire gene. In some embodiments, the inserted or substituted nucleic acid comprises a non-natural sequence. The non-natural indicator gene can be inserted into the phage genome so that it is under the control of the phage promoter. Therefore, in some embodiments, the non-natural indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification can be configured so that the indicator protein product does not include the polypeptide of the wild-type phage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present disclosure includes a method for detecting target bacteria, which includes the step of incubating a test sample with this recombinant phage.

[0090] In some embodiments, the expression of the indicator gene in the progeny phage after the host bacteria is infected produces a free soluble protein product. In some embodiments, the non-natural indicator gene is discontinuous with the gene encoding the structural phage protein, and therefore does not produce a fusion protein. In some cases, it is advantageous to use a non-fusion protein system. For example, the fusion protein needs to be first cut off by proteolysis by the indicator peptide, and then the phage particles are purified from the peptide tag (for example, HiBiT) in the reserve lysate. Different from the system using the indicator protein to fuse with the capsid protein (i.e., fusion protein), some embodiments of the present invention express soluble indicators or reporters (for example, soluble luciferase). In some embodiments, the indicator or reporter ideally do not contain phage structure. That is, the indicator or reporter is not attached to the phage structure. Therefore, the gene of the indicator or reporter is not fused with other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (as low as a single bacterium) and simplify the assay, allowing to complete the assay in less than an hour in some embodiments, rather than requiring several hours to produce a detectable fusion protein due to the need for an additional purification step for the construct. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding restrictions that may alter the conformation of the enzyme active site or access to substrates.

[0091] In other embodiments, a fusion protein comprising a marker unit is expressed in progeny phage after infection of the host bacterium. In some cases, the marker subunit gene is adjacent to a gene encoding a structural phage protein, thereby producing a fusion protein. Fusion proteins may introduce folding constraints that may alter the conformation of the enzyme active site or access to the substrate.

[0092] To maintain the advantages of non-fusion protein systems and keep the transgenic insert small, in some embodiments, a peptide or polypeptide tag (e.g., HiBiT) is fused to a small protein that can act as a stabilizing domain. This can be achieved by fusing the peptide tag (e.g., HiBiT) to a truncated form of a known larger protein, or fusing the peptide tag (e.g., HiBiT) to a known small protein. For example, small proteins include the 6.5 kDa aprotinin (encoded by 177 nucleotides) or the 14 kDa alpha lactalbumin (encoded by 372 nucleotides). In certain embodiments, there are short amino acid linkers between the domains. Even with a linker region (e.g., HiBiT with a gly-ser-gly-ser linker is 48 nucleotides long), these soluble fusion protein genes will be smaller than other photoproteins known in the art (e.g., 516 nucleotides long). ). In some embodiments, the subunit of the indicator phage encoding reporter (e.g., detectable enzyme). The reporter (indicator complex) can generate light and / or can be detected by color change. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP) or luciferase (Luc). In some embodiments, these enzymes can be used as reporters. In some embodiments, firefly luciferase is a reporter. In some embodiments, Oplophorus luciferase is a reporter. Other engineered luciferases or other enzymes that produce detectable signals can also be suitable indicator protein products.

[0093] In some embodiments, the use of soluble indicator protein products eliminates the need to remove contaminating parent phages from the lysate of infected sample cells.Using a fusion protein system, any phage used to infect sample cells will be attached with an indicator protein product and will be indistinguishable from the daughter phages that also contain the indicator protein product. Since the detection of sample bacteria relies on the detection of newly generated (de novo synthesized) indicator protein products, additional steps are required using fusion constructs to separate the old (parent) part (indicator protein) from the newly generated (daughter phage) part (indicator protein). This can be accomplished by washing infected cells multiple times before completing the phage life cycle, inactivating excess parent phages by physical or chemical means after infection, and / or chemically modifying parent phages with binding moieties (e.g., biotin), which can then be combined with and separated (e.g., by streptavidin-coated agarose beads) to achieve. However, even with all these removal attempts, when high concentrations of parental phage are used to ensure infection of a small number of sample cells, parental phage may remain, generating a background signal that can mask the detection of signals from progeny phage from infected cells.

[0094] On the contrary, for the soluble indicator protein product expressed in some embodiments of the present disclosure, it is unnecessary to purify the parent phage from the final lysate because the parent phage does not have any indicator protein product attached. Therefore, any indicator protein product present after infection must be generated from the beginning, indicating that there are infected bacteria. In order to take advantage of this benefit, the generation and preparation of the parent phage can include purifying the phage from any free indicator protein produced during the parent phage production in bacterial culture. Standard phage purification techniques can be used to purify some embodiments of the phage according to the present invention, such as sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography and dialysis or derivatization techniques (such as Amicon brand concentrators-Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as a part of the preparation of the recombinant phage of the present invention to separate the contaminating luciferase protein produced when the parent phage particles are proliferated in the bacterial host. In this way, the parent recombinant phage of the present invention is substantially free of any luciferase produced during bacterial production. Removal of residual luciferase present in phage stocks can significantly reduce the background signal observed when recombinant phage are incubated with test samples.

[0095] Standard phage purification techniques can be used to purify phage according to some embodiments of the present disclosure, such as sucrose density gradient centrifugation, cesium chloride isopycnic gradient centrifugation, HPLC, size exclusion chromatography and dialysis or derivatization techniques (e.g., Amicon brand concentrators—Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as a part of the recombinant phage preparation of the present disclosure to separate the parent phage particles from the contaminating luciferase protein produced when the phage is propagated in the bacterial host. In this way, the parent recombinant phage of the present disclosure is substantially free of any luciferase generated during production in the bacteria. Removing the residual luciferase present in the phage stock solution can significantly reduce the background signal observed when the recombinant phage is incubated with the test sample.

[0096] In some embodiments, the late promoter is a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter, or another phage promoter similar to that found in the selected wild-type phage (i.e., without genetic modification). The late gene region can be a class III gene region, and the phage can be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, a Staphylococcus or Staphylococcus aureus specific phage, or another natural phage whose genome is at least 70, 75, 80, 85, 90, or 95% homologous to T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, a Staphylococcus or Staphylococcus aureus specific phage, or has a high affinity for the RNA polymerase of the same phage that transcribes genes for structural proteins assembled into phage particles. Use of a viral late promoter can ensure optimal high-level expression of the luciferase indicator protein product. The use of a viral late promoter (e.g., a T4, T7, ViI or Saka late promoter based on a T4-, T7-, ViI- or Saka-based system) derived from, specific for or active against the original wild-type phage from which the indicator phage is derived can further ensure optimal expression of the indicator protein product. In some cases, the use of standard bacterial (non-viral / non-phage) promoters may be detrimental to expression because these promoters are typically downregulated during phage infection (so that the phage prioritizes bacterial resources for the production of phage proteins). Therefore, in some embodiments, the phage is preferably engineered to encode and express a soluble (free) indicator protein at high levels, using a position in the genome that does not limit the number of subunits expressed to the phage structural components.

[0097] The compositions of the present disclosure can include one or more wild-type or genetically modified infectious agents (e.g., phages) and one or more indicator genes. In some embodiments, the compositions can include a mixture of different indicator phages that can encode and express the same or different indicator proteins. In some embodiments, the mixture of phages includes at least two different types of recombinant phages.

[0098] Method for preparing indicator phage

[0099] The embodiment of the method for preparing indicator phage begins with selecting a wild-type phage for genetic modification. Some phage are highly specific to the target bacteria. This provides the opportunity for highly specific detection.

[0100] Therefore, the method of the present disclosure utilizes the high specificity of binding agents, associates with the infectious factors that identify and bind to specific target microorganisms, as a method of amplifying signals and detecting low-level microorganisms (e.g., single microorganisms) present in a sample. For example, infectious factors (e.g., bacteriophages) specifically recognize the surface receptors of specific microorganisms, thereby specifically infecting these microorganisms. Therefore, these infectious factors can be suitable binding agents for targeting target microorganisms. As discussed herein, bacteriophages can replicate inside bacteria to produce hundreds of progeny phages. The detection of the indicator gene product inserted into the phage genome can be used as a measure of bacteria in a sample.

[0101] Some embodiments of the present disclosure utilize the binding specificity and high-level gene expression ability of recombinant phage for rapid and sensitive targeting to infect and promote the detection of target bacteria. In some embodiments, the indicator phage is genetically modified to include a reporter gene. In some embodiments, the late gene region of the phage is genetically modified to include an indicator (reporter) gene. In some embodiments, the indicator gene is located downstream of the major capsid gene. In other embodiments, the indicator gene is located upstream of the major capsid gene. In some embodiments, the inserted gene construct also includes its own exogenous dedicated promoter to drive the expression of the indicator gene. The exogenous promoter is a supplement to any endogenous promoter in the phage genome. Since phages produce polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron of the transcript. Conventional recombinant constructs only use endogenous phage promoters to drive the inserted gene. In contrast, adding an additional promoter upstream of the reporter gene and ribosome binding site can increase gene expression by acting as a secondary start site for transcription. The complex and compact genomes of viruses often have overlapping genes in different frames, sometimes in two different directions.

[0102] Some embodiments of the method for preparing a recombinant indicator phage include selecting a wild-type phage that specifically infects a target pathogen, such as Escherichia coli, Cronobacter species, Salmonella species, Listeria species, or Staphylococcus species; preparing a homologous recombinant plasmid / vector comprising an indicator gene; transforming the homologous recombinant plasmid / vector into the target pathogen; infecting the transformed target pathogen with the selected wild-type phage, thereby causing homologous recombination to occur between the plasmid / vector and the phage genome; and isolating specific clones of the recombinant phage.

[0103] The whole bag of tricks for designing and preparing homologous recombination plasmids is known. The whole bag of tricks for using plasmids to transform bacteria is known, including heat shock, F pilus-mediated bacterial binding, electroporation, and other methods. The whole bag of tricks for separating specific clones after homologous recombination is also known. Some method embodiments described herein utilize specific strategies.

[0104] Thus, some embodiments of methods for preparing indicator phages include the following steps: selecting a wild-type phage that specifically infects a target pathogen; determining the native sequence of the late region of the selected phage genome; annotating the genome and identifying the major capsid protein gene of the selected phage; designing a sequence for homologous recombination adjacent to the major capsid protein gene, wherein the sequence comprises a codon-optimized reporter gene; incorporating the sequence designed for homologous recombination into a plasmid / vector; transforming the plasmid / vector into the target pathogen; selecting transformed bacteria; infecting the transformed bacteria with the selected wild-type phage, thereby allowing homologous recombination to occur between the plasmid and the phage genome; determining the titer of the resulting recombinant phage lysate; and performing a limiting dilution assay to enrich and isolate the recombinant phage. Some embodiments include further repeating the limiting dilution and titration steps as needed after the first limiting dilution assay until the recombinant phage represents a detectable fraction of the mixture. For example, in some embodiments, the limiting dilution and titration steps can be repeated until at least 1 / 30 of the phage in the mixture are recombinant before isolating a specific clone of recombinant phage. In some embodiments, a 1:30 recombinant: wild-type ratio is expected to produce an average of 3.2 transducing units (TU) per 96 plaques (e.g., in a 96-well plate). As previously described in U.S. Application No. 15 / 409,258, the starting ratio of recombinant to wild-type phage can be determined by performing a limiting dilution assay based on TCID50 (tissue culture infectious dose 50%). According to a Poisson distribution, a 1:30 ratio results in a 96% chance of observing at least one TU somewhere in the 96 wells.

[0105] As described herein, in certain embodiments, it may be preferred to utilize infectious agents that have been isolated from the environment to produce the infectious agents of the present disclosure. In this way, infectious agents specific for naturally derived microorganisms can be produced.

[0106] There are many known methods and commercial products for preparing plasmids. For example, PCR, site-directed mutagenesis, restriction digestion, connection, cloning and other techniques can be used in combination to prepare plasmids. Synthetic plasmids can also be ordered commercially (such as GeneWiz). Cosmids can also be used, or the CRISPR / CAS9 system can be used to selectively edit the phage genome. Some embodiments of the method for preparing recombinant indicator phages include designing a plasmid that can be easily recombined with the wild-type phage genome to produce a recombinant genome. When designing the plasmid, some embodiments include adding a codon-optimized reporter gene, such as a luciferase gene. Some embodiments also include adding an element to the upstream non-translated region. For example, when designing a plasmid recombined with the indicator phage genome, an upstream non-translated region can be added between the sequence encoding the C-terminal end of the gp23 / major capsid protein and the start codon of the indicator subunit (such as the HiBiT indicator gene). The non-translated region can include a promoter, such as T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter. The non-translated region can also include a ribosome entry / binding site (RBS), also known as the "Shine-Dalgarno sequence" of bacterial systems. One or two of these elements, or other non-translated elements, can be embedded in a short upstream non-translated region, which consists of a random sequence that contains about the same GC content as the rest of the phage genome. The random region should not include an ATG sequence because it will act as a start codon.

[0107] The MCP fragment is a part of the structural gene encoding virion proteins. Since these virion proteins are expressed at very high levels, it can be expected that any gene inserted into this region will have similar expression levels as long as late gene promoters and / or other similar control elements are used. In some cases, an indicator (e.g., HiBiT) is fused to the major capsid protein. In some embodiments, a peptide tag (i.e., HiBiT) is fused to the N-terminal of MCP. In other embodiments, a peptide tag is fused to the C-terminal of MCP.

[0108] In some embodiments, the indicator phage is genetically engineered to include an indicator gene, such as a subunit of a luciferase gene. For example, the indicator phage can be specific to a particular target bacterium, wherein the genome comprises the sequence of a HiBit gene. The recombinant indicator HiBit phage genome can further include a common promoter of T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, ViI or other late promoters. In a further embodiment, the promoter is an exogenous promoter. It is advantageous to insert an exogenous promoter to drive the expression of the indicator gene because the expression is not limited by the expression of other phage proteins (e.g., major capsid proteins).

[0109] Therefore, in the embodiment of the recombinant phage produced by recombinant generation, indicator gene or subunit gene (for example, HiBiT) are inserted in the late gene region, just downstream of the gene encoding the major capsid protein, so as to produce the recombinant phage genome comprising HiBiT gene.Construct can additionally include T7, T4, T4 sample, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, ViI or another late promoter or another suitable promoter to drive transcription and expression of luciferase gene.Construct can also include the composite non-translated region synthesized from several UTRs.This construct ensures the production of soluble luciferase so that expression is not restricted by the capsid protein quantity inherent in phage display systems.

[0110] Recombinant phage produced 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 a high-titer recombinant indicator phage stock suitable for use in detection assays. In addition, cesium chloride isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein to reduce background.

[0111] Methods for detecting bacteria using infectious agents

[0112] As described herein, in certain embodiments, the present disclosure may include methods of detecting microorganisms using infectious particles. The methods of the present disclosure may be embodied in a variety of ways.

[0113] In one embodiment, the present invention may include a method for detecting a target bacterium in a sample, comprising the steps of: incubating the sample with a bacteriophage that infects the target bacterium, wherein the bacteriophage comprises an indicator gene or a subunit of an indicator gene, such that expression of the indicator gene or subunit during phage replication after infection with the target bacterium results in the production of an indicator protein product; and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the indicator protein product is a fusion protein. In other embodiments, the indicator protein product is a soluble non-fusion protein.

[0114] In certain embodiments, assays can be performed to utilize a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using indicator phages of the present disclosure can allow for rapid detection of specific bacterial strains, such as E. coli, Cronobacter spp., Salmonella spp., Listeria spp., or Staphylococcus spp., with a total assay time of less than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 , 11.5, 12, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5 or 26.0 hours depending on the sample type, sample size and assay format. For example, the amount of time required may be slightly shorter or longer depending on the phage strain and bacterial strain to be detected in the assay, the type and size of the sample to be tested, the conditions required for target survival, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants.

[0115] In some embodiments, phage (e.g., SEA1, TSP1, TSP12, T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 phage) can be engineered to express an indicator gene or subunit during phage replication. The expression of the indicator gene is driven by a viral capsid promoter (e.g., phage T7 or T4 late promoter), resulting in high expression.

[0116] The parental phage will express the indicator protein or subunit (e.g., HiBit). Therefore, it is usually necessary to separate the parental phage from the progeny phage or remove the indicator protein or subunit from the parental phage so that the signal in the assay comes from the replication of the progeny phage during infection of the bacterial cell, rather than from the parental phage.

[0117] In some embodiments, there is no background signal from the parent phage or substantially no background signal from the parent phage. In some cases, the indicator or subunit (e.g., HiBiT) is non-luminescent or substantially non-luminescent. In a further embodiment, the indicator or subunit is removed from the parent phage before adding the substrate, so that the signal detected in the assay is definitely from the replication of the progeny phage during the bacterial cell infection period. Any method known in the art can be used to remove the indicator or subunit (e.g., HiBiT) from the parent phage. For example, a protease-cleavable linker (cleavage tag) can be cloned into the parent phage. The selection of a suitable cleavage tag depends on the selected phage. For example, the cleavage tag can be selected from 3C (PreScission) (LEVLFQ / GP), EKT (Enterokinase) (DDDDK / ), FXa (FXa Factor) (IEGR / ), TEV (Tobacco Etch Virus) (ENLYFQ / G) and thrombin (LVPR / GS). The main cleavage site of each cleavage tag is represented by " / ". Therefore, in some embodiments, the indicator phage comprises a protease cleavage site. In some embodiments, the indicator phage can comprise a fusion of an indicator subunit (peptide) and a capsid protein containing a protease cleavage site. The protease cleavage site can be recombinant, i.e., added or produced during the genetic modification process. In a further embodiment, a protease is added to the parent phage. In some embodiments, a protease is added during phage preparation to remove the indicator subunit from the parent phage, thereby producing a soluble indicator subunit (peptide). In a further embodiment, a protease is added after concentrating the phage. In some cases, a protease is added before purification to remove any residual indicator protein that may be generated when producing an infectious agent stock solution.

[0118] The selected protease is specific for the cleavage tag. The 3C, EKT, FXa, TEV, and thrombin cleavage tags are cleaved by human rhinovirus (HRV), enterokinase, factor FXa, tobacco etch virus protease, and thrombin, respectively. Each cleavage enzyme has different specificities. For example, HRV is a highly specific protease that cleaves between the Glu and Gly residues in the cleavage tag. Enterokinase is an intestinal enzyme that is commonly involved in the protease cleavage of trypsin. It cleaves after lysine (K) in the recognition sequence. Factor Xa cleaves after an Arg residue but also cleaves less frequently at secondary basic sites. Its most common secondary cleavage site is between the Gly and Arg residues in its own recognition site, although the frequency of these events is protein-specific. TEV cleaves between the Glu and Gly residues. TEV has been reported to have better specificity for its recognition site than EKT, thrombin, or factor Xa. Thrombin preferentially cleaves between Arg and Gly residues. Off-target cleavage may occur at nonspecific sites, often from contaminating proteases. To ensure maximum protein integrity, enzyme reagents must be extremely pure.

[0119] In some embodiments, the sample can be enriched prior to testing by incubation under growth-promoting conditions. In such embodiments, the enrichment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours or longer, depending on the sample type and size.

[0120] In some embodiments, the indicator phage comprises an indicator gene, and infection of a single pathogenic cell (e.g., bacteria) can be detected by an amplified signal generated by expression of the indicator gene. Thus, the method can include detecting an indicator protein produced during phage replication, wherein detection of the indicator protein indicates the presence of target bacteria in the sample.

[0121] In one embodiment, the present disclosure may include a method for detecting a target bacterium in a sample, comprising the steps of: incubating the sample with an indicator phage that infects the target bacterium, wherein the indicator phage comprises an indicator gene inserted into a late gene region of the phage, such that expression of the indicator gene during phage replication following infection of the host bacterium results in the production of an indicator protein product; incubating the indicator protein product with a detection reagent, wherein the detection reagent comprises a polypeptide complementary to the indicator protein product, and wherein the indicator protein product and its complementary polypeptide are used to indicate a complex; and detecting the indicator complex, wherein a positive detection of the indicator complex indicates the presence of the target bacterium in the sample. In some embodiments, the amount of the indicator detected corresponds to the amount of the target bacterium present in the sample.

[0122] As described in more detail herein, the methods and systems of the present disclosure can utilize a range of concentrations of parent indicator phage to infect bacteria present in a sample. In some embodiments, the indicator phage is added to the sample at a concentration sufficient to quickly find, bind, and infect target bacteria (e.g., single cells) that are present in very low numbers in the sample. In some embodiments, the phage concentration can be sufficient to find, bind, and infect the target bacteria in less than one hour. In other embodiments, these events can occur in less than two hours or less than three hours after adding the indicator phage to the sample. For example, in some embodiments, the phage concentration used in the incubation step is greater than 1x10 5 PFU / mL, greater than 1x10 6 PFU / mL or greater than 1x10 7 PFU / mL.

[0123] In certain embodiments, the infectious agent can be purified to be free of any residual indicator protein that may be generated when the infectious agent stock solution is produced. Thus, in certain embodiments, the indicator phage can be purified using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. When the infectious agent is a phage, such purification can have the additional benefit of removing phage without DNA (i.e., empty phage or "bacterial exuviae").

[0124] In some embodiments of the methods of the present disclosure, microorganisms can be detected without isolating or purifying the microorganisms from the sample. For example, in certain embodiments, a sample containing one or more target microorganisms can be directly applied to an assay container, such as a spin column, microtiter well, or filter, and the assay performed in the assay container. Various embodiments of such assays are disclosed herein.

[0125] The aliquots of the test sample can be directly distributed in the holes of the multi-well plate, indicator phage can be added, and after a period of time sufficient to infect, lysis buffer and a substrate for indicator protein (e.g., a luciferase substrate for luciferase indicator) can be added and the detection of the indicator signal can be determined. Some embodiments of the method can be carried out on a filter plate. Before infecting with indicator phage, some embodiments of the method can be carried out when the sample is concentrated or not concentrated.

[0126] For example, in many embodiments, multi-well plates are used to perform assays. The selection of plates (or any other container in which detection may be performed) may affect the detection step. For example, some plates may include a colored or white background, which may affect the detection of light emission. Generally speaking, white plates have higher sensitivity, but also produce higher background signals. Other colors of plates may produce lower background signals, but the sensitivity may also be slightly lower. In addition, one cause of background signals is light leaking from one hole to another adjacent hole. Some plates have white holes, but the rest of the plate is black. This allows high signals to be generated in the holes, but prevents light leakage between the holes, and therefore can reduce background. Therefore, the selection of plates or other assay containers may affect the sensitivity and background signals of the assay.

[0127] The methods of the present disclosure can include various other steps to increase sensitivity. For example, as discussed in more detail herein, the methods can include a step of washing the captured and infected bacteria after adding the phage but before incubation to remove excess parent phage and / or luciferase or other reporter proteins that contaminate the phage preparation.

[0128] In some embodiments, the detection of target microorganisms can be completed without the need for culture sample as a way to increase microbial population. For example, in certain embodiments, the total time required for detection is less than 26.0, 25.0, 24.0, 23.0, 22.0, 21.0, 20.0, 19.0, 18.0, 17.0, 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, 1.0 hour, 45 minutes or less than 30 minutes. It is crucial to shorten the time to obtain results for food and environmental testing of pathogens to the greatest extent possible.

[0129] In contrast to assays known in the art, the methods of the present disclosure can detect single microorganisms. Therefore, in certain embodiments, the methods can detect microorganisms (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms) of ≤10 cells present in a sample. For example, in certain embodiments, indicator phages are highly specific to specific target bacteria. In one embodiment, recombinant phages can distinguish target bacteria in the presence of other types of bacteria. In certain embodiments, recombinant phages can be used to detect a single bacterium of a particular type in a sample. In certain embodiments, recombinant phages detect 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.

[0130] Therefore, aspects of the present disclosure provide a method for detecting the microorganism in a test sample by indicating a complex. In some embodiments, when the target microorganism is an antibacterial, one or more subunits of the indicator complex can associate with an infectious agent, for example, an indicator phage. The indicator complex can react with a substrate to emit a detectable signal or can emit an intrinsic signal (such as a fluorescent protein). In some embodiments, the detection sensitivity can reveal the presence of the target microorganism of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 cells in the test sample. In some embodiments, even the target microorganism of a single cell can also produce a detectable signal. In some embodiments, the phage is a T4 sample or a ViI sample phage.

[0131] In some embodiments, the indicator protein encoded by the infectious agent can be detectable during or after the replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator parts are known in the art, and many are commercially available. In some embodiments, the indicator phage comprises an indicator gene encoding an enzyme, and the enzyme is used as an indicator protein. In other embodiments, the indicator phage comprises an indicator gene encoding a subunit of an enzyme, and the subunit of the enzyme is used as an indicator part. In some embodiments, the genome of the indicator phage is modified to encode a soluble non-fusion protein. In other embodiments, the genome of the indicator phage is modified to encode a fusion protein. In some embodiments, the indicator phage encodes a subunit (i.e., an indicator protein product) of a detectable enzyme. In some embodiments, the subunit of the detectable enzyme is hatched in the presence of a complementary subunit of the detectable enzyme so that they are reconstructed to form a functional enzyme (i.e., an indicator complex). The indicator complex can be luminescent and / or can be detectable by adding a color change of a substrate. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can be used as indicator proteins. In some embodiments, Firefly luciferase is a detectable enzyme. In some embodiments, Oplophorus luciferase is a detectable enzyme. In some embodiments, In some embodiments, the HiBiT-LgBiT complex is a detectable enzyme. In some embodiments, is a detectable enzyme. Other engineered luciferases or other enzymes that produce a detectable signal can also be used with the embodiments described in detail herein.

[0132] In some embodiments, the indicator gene encodes a subunit of a protein that can emit an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein or others). The subunit of the indicator protein (the marker subunit) can be reconstructed with the second subunit of the protein (the detection subunit) to form an indicator complex. The indicator complex can emit light and / or can be detectable by color change. In some embodiments, the indicator complex is a functional enzyme (e.g., luciferase) that interacts with a substrate to produce a signal. In some embodiments, the marker subunit is a subunit of a luciferase gene. In some embodiments, the luciferase gene is Oplophorus luciferase, firefly luciferase, Renilla luciferase, Gaussia luciferase, Lucia luciferase or an engineered luciferase, for example Rluc8.6-535 or Orange Nano-lantern.

[0133] Detection indicator can include the emission of detection light. In some embodiments, a photometer can be used to detect the reaction of an indicator (e.g., luciferase) and a substrate. The detection of RLU can be achieved by a photometer, or other machines or devices can be used. For example, a spectrophotometer, a CCD camera or a CMOS camera can detect color changes and other light emissions. Absolute RLU is very important for detection, but the signal-to-background ratio also needs to be very high (e.g., >2.0, >2.5 or >3.0) to reliably detect individual cells or a small amount of cells.

[0134] In some embodiments, the indicator phage is genetically engineered to comprise a subunit of a gene for an enzyme (e.g., luciferase). Therefore, enzyme (e.g., luciferase) is produced only when the phage specifically recognizes and infects the bacterium and subsequently reconstructs the marker subunit expressed on progeny phage with a detection subunit. In some cases, the indicator portion is expressed in the late stage of the viral life cycle. In some embodiments, the indicator is a fusion protein. In other embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to the phage structural protein that limits its copy number.

[0135] Thus, in some embodiments using indicator phage, the present disclosure includes methods for detecting a target microorganism comprising the steps of: capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phage; allowing time for infection and replication to produce progeny phage and express the indicator moiety; reconstituting the indicator moiety with a detection subunit, thereby forming an indicator complex; and detecting the progeny phage, or preferably the indicator complex, wherein detection of the indicator complex demonstrates the presence of the bacteria in the sample.

[0136] For example, in some embodiments, the test sample bacteria can be captured by being attached to the surface of the plate, or by filtering the sample through a bacteria filter (for example, 0.45 μm pore size spin filter or plate filter). In one embodiment, an infectious agent (for example, indicator phage) is directly added to the capture sample on the filter with minimum volume. In one embodiment, the microorganism captured on the filter or plate surface is washed once or repeatedly to remove excessive unbound infectious agents. In one embodiment, culture medium (for example, Luria-Bertani broth, also referred to as LB, buffered peptone water, also referred to as BPW in this article, or tryptic soy broth or tryptone soy broth, also referred to as TSB in this article) can be added for further incubation time, to allow the replication of bacterial cells and phage and the high-level expression of the gene of the coding indicator part. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage only requires sufficiently long time for a single phage life cycle. In the past, it was believed that the amplification ability using phage needed more time so that phage could replicate several cycles. According to some embodiments of the present disclosure, a single replication cycle of an indicator phage may be sufficient to facilitate sensitive and rapid detection.

[0137] In some embodiments, an aliquot of a test sample containing bacteria can be applied to a spin column, and following infection with the recombinant phage and optional washing to remove any excess phage, the amount of soluble indicator detected will be proportional to the amount of phage produced by the infected bacteria.

[0138] In some embodiments, the progeny phage are lysed prior to incubation with a detection reagent comprising a detection subunit. In some embodiments, the detection reagent also includes a substrate. For example, in a detection system utilizing the Nano-Glo HiBiT Lysis Detection System technology, the progeny phage expressing HiBiT must be lysed to bring the complementary polypeptide LgBiT into proximity with the HiBiT. When the HiBiT encounters the LgBiT, they reconstitute to form a functional luciferase.

[0139] The soluble indicator released into the surrounding liquid when the bacteria are lysed can then be measured and quantified. In one embodiment, the solution is rotated through a filter, and after adding a substrate (e.g., a luciferase substrate) for an indicator enzyme, the filtrate is collected in a new container (e.g., in a photometer) for measuring. Alternatively, the indicator signal can be measured directly on the filter.

[0140] In various embodiments, the parent indicator phage of purification does not include detectable indicator itself, because the parent phage can be purified before being used to hatch with the test sample. The expression of late (III class) genes occurs in the late stage of the viral life cycle. In some embodiments of the present disclosure, the parent phage can be purified to exclude any indicator protein (e.g., luciferase) present. In some embodiments, the expression of the indicator gene during phage replication after host bacteria infection produces a soluble indicator protein product. Therefore, in many embodiments, it is not necessary to separate the parent phage from the progeny phage before the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a phage. In one embodiment, the indicator protein is a soluble luciferase, which is released when the host microorganism is cracked.

[0141] Thus, in an alternative embodiment, an indicator substrate (e.g., a luciferase substrate) can be incubated with the portion of the sample retained on the filter or bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a conventional 96-well plate), and the substrate reaction can be detected by placing the plate directly in a luminometer.

[0142] For example, in one embodiment, the present disclosure can include a method for detecting Salmonella spp., comprising the steps of: infecting cells captured on a 96-well filter plate with a plurality of parental indicator phage that are capable of expressing luciferase upon infection; washing away excess phage; adding LB broth and allowing time for the phage to replicate and lyse specific Salmonella spp. targets (e.g., 30-120 minutes); and detecting the indicator luciferase by adding a luciferase substrate directly to the 96-well plate and measuring luciferase activity, wherein detection of luciferase activity indicates the presence of Salmonella spp. in the sample.

[0143] In another embodiment, the present disclosure may include a method for detecting Salmonella species, 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 an indicator gene upon infection; allowing time for the phage to replicate and lyse specific Salmonella species targets (e.g., 30-120 minutes); and detecting the indicator luciferase by adding a luciferase substrate directly in a 96-well plate and measuring luciferase activity, wherein detection of luciferase activity indicates the presence of Salmonella species in the sample. In such an embodiment, a capture step is not required. In some embodiments, the liquid solution or suspension can be a consumable test sample, such as a vegetable wash. In some embodiments, the liquid solution or suspension can be a vegetable wash fortified with concentrated LB broth, trypsin / tryptone soy broth, peptone water, or nutrient broth. In some embodiments, the liquid solution or suspension can be a bacterial dilution in LB broth.

[0144] In some embodiments, the reaction of the indicator protein (e.g., luciferase) with the substrate can last for 30 minutes or longer, and detection at different time points is desirable for optimizing sensitivity. For example, in embodiments using a 96-well filter plate as a solid support and luciferase as an indicator, photometer readings are taken initially and at intervals of 10 or 15 minutes until the reaction is complete.

[0145] Surprisingly, the high concentration of phage used to infect the test sample has successfully achieved detection of very low numbers of target microorganisms in a very short time. In some embodiments, the incubation of phage with the test sample only needs to be long enough for a single phage life cycle. In some embodiments, the phage concentration of this incubation step is greater than 7x10 6 , 8x10 6 , 9x10 6 , 1.0x10 7 , 1.1x10 7 , 1.2x10 7 , 1.3x10 7 , 1.4x10 7 , 1.5x10 7 , 1.6x10 7 , 1.7x10 7 , 1.8x10 7 , 1.9x10 7 , 2.0x10 7 , 3.0x10 7 , 4.0x10 7 , 5.0x10 7 , 6.0x10 7 , 7.0x10 7 , 8.0x10 7 , 9.0x10 7 or 1.0x10 8 PFU / mL.

[0146] The success of such high concentrations of phage is surprising because large amounts of phage have previously been associated with "self-lysis", which kills target cells and prevents early phage assays from producing useful signals. Cleaning up the prepared phage stock solution described herein may help alleviate this problem (e.g., by cesium chloride isopycnic density gradient ultracentrifugation) because, in addition to removing any contaminating indicator genes associated with the phage, this cleanup also removes bacterial exuviae (particles that have lost their DNA). Exuviae can lyse bacterial cells by "self-lysis", killing the cells prematurely and thus preventing the production of indicator signals. Electron microscopy has shown that crude phage lysates (i.e., before cesium chloride cleanup) may contain more than 50% exuviae. These exuviae particles may cause premature death of microorganisms by the action of many phage particles piercing the cell membrane. Therefore, exuviae particles may have caused previous problems where high PFU concentrations were reported to be harmful. In addition, very clean phage preparations allow assays to be performed without a washing step, which allows the assay to be performed without an initial concentration step. Some embodiments do include an initial concentration step, and in some embodiments, this concentration step allows for shorter enrichment incubation times.

[0147] Some embodiments of the test method may further include a confirmatory assay. Various assays are known in the art for confirming initial results, typically at a later time point. For example, the sample may be cultured (e.g., Assays, PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm initial results.

[0148] In certain embodiments, in addition to detection with infectious agents, the methods of the present disclosure are combined with the use of binding agents (e.g., antibodies) to purify and / or concentrate target microorganisms from a sample. For example, in certain embodiments, the present disclosure includes a method for detecting a target microorganism in a sample, comprising the steps of: capturing the microorganism from a sample previously on a support using a capture antibody specific for the target microorganism; incubating the sample with a recombinant indicator phage comprising an indicator gene, wherein the indicator gene encodes the first subunit of the indicator protein, thereby producing a certain amount of progeny phage expressing the first subunit; lysing a certain amount of progeny phage; incubating the lysed progeny phage in the presence of a detection reagent, wherein the detection reagent comprises the second subunit of the indicator protein, thereby allowing the first subunit and the second subunit to be reconstituted to form an indicator protein complex; and detecting the indicator protein complex, wherein a positive detection of the indicator protein complex indicates the presence of a specific target bacterium in the sample. In some embodiments, synthetic phages are designed to optimize the desired traits for pathogen detection assays. In some embodiments, bioinformatics and previous genetic modification analysis are used to optimize the desired traits. For example, in some embodiments, the gene encoding the phage tail protein can be optimized to recognize and bind to a specific type of bacteria. In other embodiments, the gene encoding the phage tail protein can be optimized to recognize and bind to an entire bacterial genus or a specific group of species within a genus. In this way, phage can be optimized to detect a wider or narrower group of pathogens. In some embodiments, synthetic phage can be designed to improve the expression of reporter genes. Additionally and / or alternatively, in some cases, synthetic phage can be designed to increase the amount of phage lysis to improve detection.

[0149] In some embodiments, the stability of the phage can be optimized to improve shelf life. For example, the solubility of the enzyme can be increased to increase the subsequent stability of the phage. Additionally and / or alternatively, the thermostability of the phage can be optimized. Thermostable phage better retain functional activity during storage, thereby increasing shelf life. Thus, in some embodiments, thermostability and / or pH tolerance can be optimized.

[0150] Systems and kits of the present disclosure

[0151] In some embodiments, the present disclosure includes a system (e.g., an automated system or a test kit) comprising components for performing the methods disclosed herein. In some embodiments, an indicator phage is included in a system or test kit according to the present disclosure. The methods described herein may also utilize such an indicator phage system or test kit. In view of the minimum amount of reagents and materials required for performing the methods, some embodiments described herein are particularly suitable for automation and / or test kits. In certain embodiments, each component of the test kit may include a separate unit that can be delivered from a first site to a second site.

[0152] In some embodiments, the present disclosure includes a system or kit for rapid detection of target microorganisms in a sample. In certain embodiments, the system or kit may include a component for incubating the sample with an infectious agent specific to the target microorganism, wherein the infectious agent includes an indicator gene and a component for detecting an indicator protein. In some embodiments of both the system and the kit of the present disclosure, the infectious agent is a recombinant bacteriophage that infects the target bacteria, and the recombinant bacteriophage includes an indicator gene encoding a peptide or polypeptide subunit of an indicator protein. In some embodiments, the indicator gene inserted into the late gene region of the phage is an indicator portion so that the expression of the indicator gene during phage replication after infecting the host bacteria produces a subunit of an indicator protein product. Therefore, some systems also include a detection reagent, wherein the detection reagent includes a polypeptide subunit of an indicator protein, wherein the polypeptide subunit is reconstructed with the peptide subunit to form an indicator protein complex, and a substrate for reacting with the indicator protein complex to detect the indicator protein complex. In addition, some systems also include a component for capturing the target microorganism on a solid support.

[0153] In other embodiments, the present disclosure includes methods, systems, or kits for rapidly detecting a target microorganism in a sample, comprising an infectious agent component specific for the target microorganism, wherein the infectious agent comprises an indicator moiety, and components for detecting the indicator protein. In certain embodiments, the recombinant bacteriophage is highly specific for a particular bacterium.

[0154] 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 an indicator moiety, wherein the amount of the 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 activity.

[0155] In some systems and / or kits, the same component can be used in multiple steps. In some systems and / or kits, the steps are automated or controlled by the user via computer input and / or wherein a liquid handling robot performs at least one step.

[0156] Therefore, in certain embodiments, the present disclosure may include a system or kit for rapidly detecting a target microorganism in a sample, comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent comprises an indicator portion; a component for capturing the microorganism from the sample on a solid support; a component for washing the captured microorganism sample to remove unbound infectious agents; and a component for detecting the indicator portion. In some embodiments, the same component can be used for the steps of capturing and / or incubating and / or washing (e.g., a filter component). Some embodiments additionally include a component for determining the amount of the target microorganism in the sample, wherein the amount of the indicator portion detected corresponds to the amount of the microorganism in the sample. Such a system may include various embodiments and sub-embodiments similar to the above-mentioned method for rapidly detecting microorganisms. 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 thereof).

[0157] In some embodiments, the system can include components for separating the target microorganism from other components in a sample.

[0158] In one embodiment, the present disclosure includes a system or kit comprising components for detecting a target microorganism, comprising: a component for separating at least one microorganism from other components in a sample; a component for infecting 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 progeny infectious agents or detecting soluble proteins encoded and expressed by infectious agents with higher sensitivity, wherein detection of an infectious agent or a protein product of an infectious agent or a subunit thereof indicates the presence of a microorganism in the sample. In some embodiments, the system or kit may further include a component for reconstructing a subunit of a protein product of an infectious agent to form a detectable indicator protein complex. The infectious agent may include a bacteriophage carrying a HiBiT indicator gene.

[0159] In other embodiments, the present disclosure may include a kit for rapidly detecting a target microorganism in a sample, the system comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent comprises a subunit of an indicator protein; a component for capturing the microorganism from the sample on a solid support; a component for washing the captured microbial sample to remove unbound infectious agents; and a component for detecting the indicator protein (i.e., a detection reagent). In some embodiments, the detection reagent comprises a polypeptide subunit of the indicator protein, wherein the polypeptide subunit is reconstructed with the peptide subunit to form an indicator protein complex, and a substrate for reacting with the indicator protein complex to detect the indicator protein complex. The same components can be used for the steps of capturing and / or incubating and / or washing. Some embodiments additionally include a component for determining the amount of the target microorganism in the sample, wherein the amount of the indicator protein complex detected corresponds to the amount of the microorganism in the sample. Such a kit may include various embodiments and sub-embodiments similar to the above-mentioned method for rapidly detecting microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0160] In some embodiments, the kits can include components for capturing target microorganisms.

[0161] In some embodiments, the kits can include components for separating the target microorganism from other components in a sample.

[0162] The systems and kits of the present disclosure include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of devices suitable for performing the methods. The components need not be integrally connected or positioned with each other in any particular manner. The present disclosure encompasses any suitable arrangement of the components relative to each other. For example, the components do not need to be in the same room. However, in some embodiments, the components are connected to each other in an integral unit. In some embodiments, the same component can perform multiple functions.

[0163] Computer system and computer readable medium

[0164] The system or any component thereof as described in the present technology can be embodied in the form of a computer system. Typical examples of computer systems include general-purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit components, and other devices or device arrangements capable of implementing the steps constituting the methods of the present technology.

[0165] A computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may also include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may also include a storage device. The storage device may be a hard drive or a removable storage drive, such as a floppy disk drive, an optical disk drive, etc. The storage device may also be other similar tools 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 via an I / O interface. The communication unit allows data to be transmitted to and received from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that enables the computer system to connect to databases and networks such as a LAN, MAN, WAN, and the Internet. Thus, the computer system can facilitate user input through the input device and access to the system through the I / O interface.

[0166] A computing device will typically include an operating system that provides executable program instructions for the general management and operation of the computing device, and will typically include a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) storing instructions that, when executed by a processor of the server, enable the computing device to perform its intended functions. Suitable implementations of the operating system and general functionality of a computing device are known or commercially available and are readily implementable by one of ordinary skill in the art, particularly in light of the disclosure herein.

[0167] A computer system executes a set of instructions stored in one or more memory elements in order to process input data. Memory elements can also store data or other information as needed. Memory elements can be in the form of information sources or physical memory elements present in a processing machine.

[0168] The environment can include various data storage devices and other storage devices and storage media as described above. These can reside in various locations, such as on a storage medium local to one or more computers (and / or residing therein), or on a storage medium away from any or all computers on a network. In a specific set of embodiments, information can reside in a storage area network ("SAN") familiar to those skilled in the art. Similarly, any necessary files for performing functions attributed to a computer, server, or other network device can be stored locally and / or remotely as appropriate. In the case where the system includes a computing device, each such device can include hardware elements that can be electrically coupled by a bus, including, for example, at least one central processing unit (CPU), at least one input device (such as a mouse, keyboard, controller, touch screen, or keypad), and at least one output device (such as a display device, printer, or speaker). Such a system can 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 memory cards, etc.

[0169] Such a device may also include a computer-readable storage medium reader, a communication device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.) and a working memory as described above. The computer-readable storage medium reader may be connected to or configured to receive a computer-readable storage medium, which represents a remote, local, fixed and / or removable storage device and a storage medium for temporarily and / or more permanently containing, storing, transmitting and retrieving computer-readable information. The system and various devices will typically also include a plurality of software applications, modules, servers or other elements located within at least one working memory device, including an operating system and applications, such as a client application or a web browser. It should be understood that alternative embodiments may have many variations different from the above-described embodiments. For example, customized hardware may also be used and / or specific elements may be implemented in hardware, software (including portable software, such as applets), or both. In addition, connections to other computing devices such as network input / output devices may be employed.

[0170] Non-transitory storage media and computer-readable media for containing code or portions of code may include any suitable media known or used in the art, including storage media and communication media, such as, but not limited to, volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing and / or transmitting 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 required information and can be accessed by system devices. Based on the disclosure and teachings provided herein, those of ordinary skill in the art will understand other ways and / or methods of implementing various embodiments.

[0171] Computer-readable media can include, but are not limited to, electronic, optical, magnetic or other storage devices that can provide computer-readable instructions to the 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 such as NAND flash memories or NOR flash memories, ASICs, configured processors, optical memories, magnetic tapes or other magnetic memories, or any other medium from which a computer processor can read instructions. In one embodiment, a computing device can include a single type of computer-readable medium, such as a random access memory (RAM). In other embodiments, a computing device can include two or more types of computer-readable media, such as a random access memory (RAM), a disk drive, and a cache. A computing device can communicate with one or more external computer-readable media, such as an external hard drive or an external DVD or Blu-ray drive.

[0172] As discussed above, embodiment includes processor, it is configured to carry out computer executable program instruction and / or access the information stored in memory.Instruction can comprise processor-specific instruction generated by compiler and / or interpreter from the code written in any suitable computer programming language, including for example C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript and ActionScript (Adobe Systems, Mountian View, Calif.).In one embodiment, computing device comprises single processor.In other embodiments, equipment comprises two or more processors.Such processor can comprise microprocessor, digital signal processor (DSP), application specific integrated circuit (ASIC), field programmable gate array (FPGA) and state machine.Such processor can also comprise programmable electronic device such as PLC, programmable interrupt controller (PIC), programmable logic device (PLD), programmable read-only memory (PROM), electronic programmable read-only memory (EPROM or EEPROM) or other similar devices.

[0173] Computing device includes a network interface. In some embodiments, the network interface is configured to communicate via a wired or wireless communication link. For example, the network interface can allow communication on a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface can allow communication on a network via, for example, CDMA, GSM, UMTS or other cellular communication networks. In some embodiments, the network interface can allow point-to-point connection with another device, such as via a universal serial bus (USB), 1394 FireWire, serial or parallel connection or similar interface. Some embodiments of suitable computing devices may include two or more network interfaces for communicating on one or more networks. In some embodiments, computing device may include a data storage device in addition to or instead of a network interface.

[0174] Some embodiments of suitable computing devices may include or communicate with a plurality of external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, audio speakers, one or more microphones, or any other input or output device. For example, the computing device may communicate with various user interface devices and displays. The display may use any suitable technology, including but not limited to LCD, LED, CRT, etc.

[0175] The instruction set executed by the computer system may include various commands that instruct the processing machine to perform a specific task (e.g., the steps constituting the method of the present technology). The instruction set may be in the form of a software program. In addition, as in the present technology, the software may be in the form of a collection of separate programs, a program module having a larger program, or a portion of a program module. 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 requests made by another processing machine.

[0176] Although the present disclosure has been disclosed with reference to certain embodiments, many modifications, variations and changes to the described embodiments are possible without departing from the scope and spirit of the present disclosure as defined in the appended claims. Therefore, it is intended that the present disclosure not be limited to the described embodiments, but that it have the full scope defined by the language of the appended claims and their equivalents. Example

[0177] The methods are described in the following examples, and the results described in the following examples demonstrate that small numbers of cells, even single bacteria, can be detected, with results obtained in a shortened time.

[0178] Example 1. Expression of soluble TSP12.Hibit in Salmonella

[0179] The soluble HiBiT TSP12 construct was cloned into the pUC57.Amp plasmid to generate TSP12.HR.HiBiT. The plasmid was transformed into Salmonella bongori ATCC 43975 and transformants were selected on LB carbenicillin selective agar. Well-isolated colonies were selected and inoculated into selective broth and cultured for 2 hours. After incubation, 5 μL of culture was assayed using the HiBiT Lytic Assay Kit according to the manufacturer's instructions to determine HiBiT expression. The detected signal range was 11,000-100,000 RLU / 5uL culture (Table 1). The plasmid contains all the necessary elements (promoter / RBS and coding sequence) for soluble HiBiT expression in Salmonella bongori 43975.

[0180] Table 1. HiBiT signals of Salmonella bongori transformants.

[0181]

[0182] The Salmonella bongori transformant with the highest HiBit signal was then infected with wild-type TSP12 at a multiplicity of infection (MOI) of 0.1. Phage lysates were clarified, filtered, and buffer exchanged. Serial dilutions of phage and phage with host cells were performed. The serial dilutions were then analyzed using the TU50 assay and the PFU titer was determined (Table 2).

[0183] Table 2. Soluble TSP12.HiBit homologous recombination screening

[0184]

[0185]

[0186] Example 2. TSP12.MCP-PS-HiBit expression in Salmonella

[0187] The TSP12 major capsid protein (MCP) HiBiT fusion construct was cloned into the pUC57.Amp plasmid. Cloning and sequence verification were performed using GeneWiz. The plasmid was reconstructed and lyophilized. The reconstructed, lyophilized plasmid was then transformed into Salmonella bongori ATCC 43975. Transformants were cultured and selected on LB carbenicillin selective agar. Isolated colonies were selected and inoculated into selective broth and incubated at 37°C for 2 hours. After incubation, 5uL of culture was assayed using the HiBiTLytic assay kit according to the manufacturer's instructions to determine HiBiT expression. The HiBiT signal detected from the transformant was approximately 800RLU / 5uL culture (Table 3). This plasmid does not contain all the necessary elements for effective MCP-HiBiT expression in Salmonella bongori ATCC 43975. In order for the HiBit fusion protein to be fully expressed, the plasmid must be recombined with the TSP12 phage.

[0188] Table 3. HiBiT signals of S. bongori transformants.

[0189]

[0190]

[0191] The Salmonella bongori transformant with the highest HiBit signal was then infected with wild-type TSP12 at a multiplicity of infection (MOI) of 0.1. Phage lysates were clarified, filtered, and buffer exchanged. Serial dilutions of phage and phage with host cells were performed. The serial dilutions were then analyzed using the TU50 assay and the PFU titer was determined (Table 4). Recombinant phage was detected at 20-80 times the background signal (cell-free control). The signal was dose-dependent and increased with increasing phage number.

[0192] Table 4. TSP12.MCP-PS-HiBit homologous recombination screening

[0193]

[0194] Example 3. Homologous recombination constructs

[0195] Homologous recombination products Figure 4 described in. Parental phage represents the wild-type input phage used for recombination. Fusion protein recognizes the protein of interest (POI) of the fusion partner used for HiBiT. HR donor plasmid provides the name of the plasmid transformed into the HR host. Vector backbone provides the name of the vector used to clone the HR region (the vector provided by GENEWIZ). GENEWIZ clone ID is a unique identifier used by GENEWIZ to track clones and report QC data. Cleavage site indicates which protease cleavage site is used to connect HiBiT and the fusion partner. HR host / strain indicates the species and strain used in recombination and isolate infection. Phage batch indicates whether the phage was completely isolated and it will be stored (NA) or processed for large-scale production (phage batch #). Incomplete means that a) the recombinant is unstable or b) it cannot be completely isolated or c) no recombination was attempted.

[0196] Example 4. Optimization of HiBiT Assay

[0197] Samples were added to microtiter assay wells. According to the HiBiT Lysis Kit manufacturer's instructions, each microtiter assay well containing sample received the components listed in Table 5. The reaction was mixed for 10 minutes by nutating before reading on a luminometer. Our preliminary experiments with transformed HR hosts and HR-infected lysates showed a relatively high background signal (~500 RLU) in culture medium alone.

[0198] Table 5. HiBiT assay components

[0199]

[0200] Subsequent experiments determined that the elevated background was attributable to the culture medium containing the sample. No elevated background levels were detected in the assays using SM buffer, water, or phage in SM buffer. To identify the source of the high background, several culture media were serially diluted 2-fold in sterile d-water, and 100 uL of each was used in the above assays and reported in Table 6.

[0201] Table 6. Culture medium background signal (RLU)

[0202]

[0203] In addition, several media components were dissolved in sterile water at two times (2X) the recommended concentration, serially diluted 2-fold and subjected to the HiBiT assay as described above (Table 7).

[0204] Table 7. 2X Medium Background Signal (RLU)

[0205]

[0206] The results show that all tested culture media have high background, and both BHI and tryptone (Sigma) have very high background (2000-6000RLU). Even if the culture media / components are diluted 8 times, the signal will appear saturated. An experiment was conducted to identify which component of the Lytic kit assay combined with BHI in the absence of HiBiT is the cause of the very high RLU. Table 8 shows that the LgBiT component interacts with BHI and produces a high RLU background in the presence of furimazine substrate. It should also be noted here that the real background of LgBiT and furimazine in the separate buffer is also as high as 110-180RLU.

[0207] Table 8. Background signal (RLU) of culture medium components

[0208]

[0209] To reduce the high background associated with LgBiT, medium, and substrate alone, the concentration of LgBiT was reduced 5-10 fold (Table 9).

[0210] Table 9 shows that when LgBiT was reduced to 0.05 μL / reaction, the background signal decreased approximately 6-fold compared to the manufacturer's recommended 0.5 μL / reaction. For all subsequent HiBiT assays, the LgBiT component was reduced to at least 0.1 μL / reaction to mitigate background RLU associated with the culture medium.

[0211] Table 9. LgBiT and culture medium background signals

[0212]

[0213] Example 5. Recombinant phage production

[0214] Recombinant phage expressing soluble HiBiT or HiBiT tags fused to structural proteins are generated by standard infection of host bacteria containing homologous recombination donor plasmids. The homologous recombination (HR) plasmids are transformed into the preferred host bacteria by electroporation. Transformants containing the plasmid are selected by growth on antibiotic selective plates. The host is grown to early to mid-logarithmic phase under selective pressure, and HiBiT activity is measured in the culture.

[0215] Bacterial cells that survive selection and show HiBiT activity are then used as hosts for HR infection. Logarithmic cells are diluted to approximately 1.0E+07 cells / mL in medium containing antibiotics. Natural / wild-type phage is added at an MOI of approximately 0.05-1.0, and the infection is incubated with shaking at the preferred temperature for 3-5 hours. After incubation, any remaining bacterial host cells and cell debris are precipitated by centrifugation at 5000xg for 5 minutes. The lysate is collected and filtered through a 0.45 micron filter to eliminate any remaining host cells.

[0216] Phage lysates were then prepared for endpoint dilution and plaque assays to determine estimated recombinant / plaque titers. The phage lysates were buffer exchanged to remove all unincorporated / host-derived HiBiT by passing the lysate through a 100,000 Dalton molecular weight cutoff spin filter with an additional 4 volumes of wash buffer. The resulting phage were suspended in 0.5 mL of buffer / medium and eight 10-fold dilutions were made in medium.

[0217] Then, by using TU 50 Phage titers were determined by titrating HR lysates of HiBiT recombinants using the TU assay (transduction units 50%). Wells that did not show HiBiT activity above background (phage and medium only) were scored as negative, and wells with 3X background RLU were scored as positive. 50 The titer was calculated based on the Reed-Muench method. The same serial dilutions were titrated by plaque assay. 50 Titers were compared with plaque assay titers to determine the recombinant / total phage titer ratio.

[0218] If the recombinant / total phage titer ratio is less than 1 / 30, perform limiting dilution enrichment until the ratio is equal to or higher than 1 / 30. Perform limiting dilution enrichment by diluting the lysate to contain 1-10 transducing units in 5 mL of culture medium. Then add the natural host bacteria to the diluted phage at a low MOI and distribute it among the wells of a 96-well culture plate. Incubate the phage infection at 25-37°C for 3-16 hours. Perform the HiBiT assay on 10% of each well, and perform the phage titer assay as described above on the top positive wells. Perform continuous limiting dilution enrichment until the recombinant / total phage titer ratio is less than 1 / 30.

[0219] Once a favorable recombinant / total titer ratio is achieved, plaque isolation of recombinant phage is performed. Individual plaques are isolated and screened for HiBiT activity. Positive plaques are passaged at least three times to achieve purity. Fully isolated recombinants are generated by large-scale infection and purified by cesium or sucrose gradient centrifugation.

[0220] Example 6. Phage concentration optimization of TSP1.sHiBiT (TSP1 with soluble unfused HiBiT monomer)

[0221] Salmonella typhimurium (ATCC 19585) was cultured in TSB at 37°C for 16-18 hours. The cells were diluted to 10, 20, 50, 100, 1000, 10,000, and 100,000 CFU / mL. 100 μL of the diluted cells was pipetted to generate 1, 2, 5, 10, 100, 1000, and 10,000 CFU / well in a white 96-well plate. 10 μL of TSP1.sHiBit phage was added at 1.2 x 10 4 to 10 8 pFU / mL was added to each well (Table 10).

[0222] Table 10. HiBiT phage assay plate layout

[0223] Phage / mL <![CDATA[10 4 P / mL]]> <![CDATA[10 5 P / mL]]> <![CDATA[10 6 P / mL]]> <![CDATA[10 7 P / mL]]> <![CDATA[10 8 P / mL]]> A 0 0 0 0 0 B 1 1 1 1 1 C 2 2 2 2 2 D 5 5 5 5 5 E 10 10 10 10 10 F 100 100 100 100 100 G 1000 1000 1000 1000 1000 H 10000 10000 10000 10000 10000

[0224] The plate was incubated at 37°C for 2 or 3 hours. 50 μL of the master mix (50 μL of assay buffer (NanoGlo HiBiT buffer), 1 μL of NanoGlo HiBit substrate, and 0.1 μL of LgBiT protein) was added to each well, incubated at room temperature on a nutator, and read for 1 second in the GloMax / Navigator. The signal / background values for the 2-hour incubation are shown in Figure 5 The signal / background values for the 3-hour incubation are shown in Figure 6 Medium. An incubation time of 3 hours had an improved signal / background ratio.

[0225] Example 7. Optimization of Phage Concentration of TSP12.sHiBiT and TSP12 Expressing Soluble HiBiT Peptide

[0226] Salmonella bongori (ATCC 43975) was cultured in TSB at 37°C for 16-18 hours. The cells were diluted to 10, 20, 50, 100, 1000, 10,000, and 100,000 CFU / ml. 100 μL of the diluted cells was pipetted to generate 1, 2, 5, 10, 100, 1000, and 10,000 CFU / well in a white 96-well plate. 10 μL of TSP12.sHiBit phage was added at 1.2 x 10 4 to 10 8PFU / mL was added to each well (Table 10). The plate was incubated at 37°C for 2 hours or 4 hours. 50 μL of the premix (50 μL of assay buffer (NanoGlo HiBiT buffer), 1 μL of NanoGlo HiBit substrate, and 0.1 μL of LgBiT protein) was added to each well, incubated at room temperature on a nutator, and read for 1 second in the GloMax / Navigator. The signal / background value for the 4-hour incubation is shown in Figure 7 The signal / background values for the 3-hour incubation are shown in Figure 8 Medium. An incubation time of 4 hours had an improved signal / background ratio.

[0227] Example 8. Optimization of phage concentration of TSP12.HiBiT-PS-Soc

[0228] Salmonella bongori (ATCC 43975) was cultured in TSB at 37°C for 16-18 hours. The cells were diluted to 10, 20, 50, 100, 1000, 10,000, and 100,000 CFU / ml. 100 μL of the diluted cells was pipetted to generate 1, 2, 5, 10, 100, 1000, and 10,000 CFU / well in a white 96-well plate. 10 μL of TSP12.HiBit-PS-Soc phage was added at 1.2 x 10 4 to 10 8 PFU / mL was added to each well (Table 10). The plate was incubated at 37°C for 2 hours. 50 μL of the premix (50 μL of assay buffer (NanoGlo HiBiT buffer), 1 μL of NanoGlo HiBit substrate, and 0.1 μL of LgBiT protein) was added to each well, incubated at room temperature on a nutator, and read for 1 second in the GloMax / Navigator. The signal / background value for the 2-hour incubation is shown in Figure 9 middle.

[0229] Example 9. Detection Level of TSP12.HiBiT-PS-Soc

[0230] Salmonella bongori (ATCC 43975) was cultured in TSB at 37°C for 16-18 hours. The cells were diluted to 10, 20, 50, 100, 1000, 10,000, and 100,000 CFU / ml. 100 μL of the diluted cells was pipetted to generate 1, 2, 5, 10, 100, 1000, and 10,000 CFU / well in a white 96-well plate. 10 μL of TSP12-HTS or TSP12-HPS phage was added at 1.2 x 10 7PFU / mL was added to each well (Table 10). The plate was incubated at 37°C for 2 hours. 50 μL of the premix (50 μL of assay buffer (NanoGlo HiBiT buffer), 1 μL of NanoGlo HiBit substrate, and 0.1 μL of LgBiT protein) was added to each well and incubated on a nutator at room temperature for 10 minutes, followed by a 1-second read in the GloMax / Navigator. The signal / background value is displayed at Figure 10 middle.

Claims

1. A kit for detecting target bacteria in a sample, the kit comprising a recombinant indicator phage, the recombinant indicator phage comprising an indicator gene inserted into the phage genome, wherein the indicator gene encodes a first peptide or polypeptide subunit of an indicator protein, and A detection reagent comprising a second polypeptide subunit of an indicator protein, wherein the second polypeptide subunit reconstitutes with the first peptide or polypeptide subunit to form an indicator protein complex, and A substrate is used for reacting with the indicator protein complex to detect the indicator protein complex.

2. The kit of claim 1, wherein the recombinant indicator phage further comprises a protease cleavage site.

3. The kit of claim 1 or 2, wherein the indicator protein complex is luciferase.

4. The kit of claim 1 or 2, wherein the recombinant indicator phage further comprises an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a phage late gene promoter and a ribosome entry site.

5. The kit of claim 1 or 2, wherein the indicator gene is inserted into the late gene region of the phage genome. The kit according to claim 1 , further comprising a lysis buffer.

7. The kit according to claim 1 or 2, further comprising a component for capturing target bacteria.

8. The kit according to claim 1 or 2, further comprising a component for separating target bacteria from other components in a sample.

9. A method for detecting a target bacterium in a sample for non-diagnostic purposes, comprising: incubating the sample with a recombinant indicator phage comprising an indicator gene, wherein the indicator gene encodes a first subunit of an indicator protein, thereby producing a certain amount of progeny phage expressing the first subunit; Lysing bacteria in the sample to release a certain amount of progeny phage and first subunit; incubating the lysed progeny phage in the presence of a detection reagent comprising the second subunit of the indicator protein, thereby allowing the first subunit and the second subunit to reconstitute to form an indicator protein complex; and The indicator protein complex is detected, wherein positive detection of the indicator protein complex indicates the presence of the target bacteria in the sample.

10. The method of claim 9, wherein the sample is a food sample, an environmental sample, a water sample, or a commercial sample.

11. The method of claim 9 or 10, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a sample of a standard size for food safety industry.

12. The method of claim 10, wherein the food sample comprises meat, fish, vegetables, eggs, dairy products, dry food, or infant formula.

13. The method of claim 9 or 10, wherein the sample is first incubated under conditions favorable for growth for an enrichment period of less than 24 hours.

14. The method of claim 9 or 10, wherein the total time to result is less than 26 hours.

15. The method of claim 9 or 10, wherein the ratio of signal to background generated by detection of the indicator protein complex is at least 2.

0.

16. A system for detecting target bacteria, the system comprising a recombinant indicator phage, the recombinant indicator phage comprising an indicator gene encoding a first peptide or polypeptide subunit of an indicator protein, and A detection reagent comprising a second polypeptide subunit of an indicator protein, wherein the second polypeptide subunit reconstitutes with the first peptide or polypeptide subunit to form an indicator protein complex, and A substrate is used for reacting with the indicator protein complex to detect the indicator protein complex.

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