Devices and methods for detecting microorganisms using recombinant reproduction-deficient indicator bacteriophage

Abstract

To provide compositions, methods, kits and systems for rapid detection of microorganisms using a reproduction-deficient indicator bacteriophage.SOLUTION: The specificity of such reproduction-deficient indicator bacteriophage for binding and infecting particular microorganisms of interest allows targeted and sensitive detection of a microorganism of interest. An exemplary embodiment of the present disclosure is a composition comprising at least two recombinant phages, each comprising an indicator gene in a late gene region of the genome of the phage, where the recombinant phages are reproduction-deficient, and where the recombinant phages are capable of specifically infecting one or more microorganisms of interest.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Incorporation by Reference This application claims priority to U.S. Provisional Patent Application No. 62 / 891,701, filed August 26, 2019. The disclosures of the following U.S. patent applications are hereby incorporated by reference in their entireties: U.S. Patent Application No. 16 / 247,490, filed January 14, 2019; U.S. Patent Application No. 16 / 247,486, filed January 14, 2019; U.S. Patent Application No. 16 / 298,695, filed March 11, 2019; U.S. Provisional Patent Application No. 62 / 640,793, filed March 9, 2018; U.S. Provisional Patent Application No. 62 / 798,980, filed January 30, 2019; U.S. Patent Application No. 13 / 773,339, filed February 21, 2013; U.S. Patent Application No. 13 / 773,339, filed February 18, 2015; U.S. Provisional Patent Application No. 14 / 625,481, filed September 13, 2016, U.S. Provisional Patent Application No. 15 / 263,619, filed January 18, 2017, U.S. Provisional Patent Application No. 62 / 616,956, filed January 12, 2018, U.S. Provisional Patent Application No. 62 / 628,616, filed February 9, 2018, U.S. Provisional Patent Application No. 62 / 661,739, filed April 24, 2018, U.S. Provisional Patent Application No. 62 / 640,793, filed March 9, 2018, and U.S. Provisional Patent Application No. 62 / 798,980, filed January 30, 2019.

[0002] FIELD OF THE INVENTION The present disclosure relates to methods, devices and systems for the detection of microorganisms of interest using recombinant infections agents. [Background technology]

[0003] background There is a strong interest in improving the speed and sensitivity of detecting bacteria, viruses, and other microorganisms in living organisms, food, water, and clinical samples. Microbial pathogens can cause substantial morbidity among humans and domestic animals, as well as enormous economic losses. In view of the outbreaks of life-threatening or fatal diseases caused by the ingestion of food contaminated with certain microorganisms, such as Staphylococcus spp., Escherichia coli, or Salmonella spp., the detection of microorganisms is a high priority for the Food and Drug Administration (FDA) and the Centers for Disease Control (CDC).

[0004] Traditional microbiological tests for bacterial detection rely on nonselective and selective enrichment cultures, followed by plating on selective media and further testing to confirm suspect colonies. Such procedures can require several days. Various rapid methods have been researched and implemented to reduce the time required. However, current methods for reducing the time required have weaknesses. For example, techniques involving direct immunoassays or gene probes generally require an overnight enrichment step to achieve adequate sensitivity and therefore lack the ability to provide same-day results. Polymerase chain reaction (PCR) tests also involve an amplification step and are therefore capable of both very high sensitivity and selectivity; however, the sample size that can be economically subjected to PCR testing is limited. Dilute bacterial suspensions that can be subjected to PCR do not contain cells, and therefore purification and / or lengthy enrichment steps are still required.

[0005] The time required for traditional biological enrichment depends on the growth rate of the target bacterial population in the sample, the effect of the sample matrix, and the required sensitivity. In fact, the most sensitive methods use overnight incubation, requiring approximately 24 hours in total. Due to the time required for culture, these methods can require up to three days, depending on the organism to be identified and the source of the sample. This delay is generally inadequate because it allows contaminated food or water or other products to enter livestock or humans. In addition, with the rise of antibiotic-resistant bacteria and biodefense considerations, the rapid identification of bacterial pathogens in water, food, and clinical samples has become a critical priority worldwide. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need for faster, simpler and more sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms. [Means for solving the problem]

[0007] overview Embodiments of the present disclosure include devices, compositions, methods, apparatus, systems, and kits for the detection of microorganisms, including but not limited to bacteria. The present disclosure can be embodied in a variety of ways. Some exemplary embodiments of the present application are described below.

[0008] An exemplary embodiment of the present disclosure is a recombinant phage comprising an indicator gene in the late gene region of the phage genome, the recombinant phage being reproductively defective and capable of specifically infecting a microorganism of interest. In some embodiments, the recombinant bacteriophage is reproductively defective due to an alteration in a late gene required for virion assembly. In some embodiments of the recombinant bacteriophage, the indicator gene is inserted into the late gene sequence of the recombinant phage, rendering the late gene non-functional and rendering the recombinant phage reproductively defective. In some embodiments of the recombinant bacteriophage, the indicator gene replaces at least a portion of the late gene sequence of the recombinant phage, rendering the recombinant phage reproductively defective, the late gene being required for virion assembly. The recombinant phage is derived from a phage specific for E. coli, Salmonella, Listeria, or Staphylococcus. In some embodiments, the recombinant phage is derived from a phage specific for E. coli. In other embodiments, the recombinant phage is derived from a phage specific for Salmonella. In some embodiments of the recombinant bacteriophage, the late genes are required for virion assembly.

[0009] An exemplary embodiment of the present disclosure is a composition comprising at least two recombinant phages, each comprising an indicator gene in the late gene region of the phage genome, wherein the recombinant phages are reproduction-deficient and capable of specifically infecting one or more target microorganisms. In some embodiments of the composition, each of the at least two recombinant phages comprises a different indicator gene. In some embodiments of the composition, each of the at least two recombinant phages is capable of specifically infecting a different target microorganism. In some embodiments of the composition, the at least two recombinant phages are capable of infecting multiple target microorganisms. In some embodiments of the composition, the multiple target microorganisms comprise at least two different categories of bacteria. In some embodiments of the composition, the at least two different categories of bacteria comprise one or more of at least two different genera of bacteria, at least two different species of bacteria, at least two different strains of bacteria, or at least two different serotypes of bacteria.

[0010] An exemplary embodiment of the present disclosure is a method for preparing a recombinant phage. Such a method can include the steps of selecting a parent phage that specifically infects a target microorganism, modifying the gene of the parent phage to generate a recombinant, reproduction-deficient phage, transforming an engineered strain of the target microorganism capable of expressing the product of the mutated gene in the reproduction-deficient phage with a homologous recombination (HR) plasmid containing an indicator gene and HR sequences flanking the indicator gene and homologous to a desired sequence in the parent phage, infecting the transformed target microorganism with the parent phage or reproduction-deficient parent phage to allow HR to occur between the HR plasmid and the genome of the parent phage or recombinant reproduction-deficient phage, and isolating specific clones of recombinant phage that are reproduction-deficient and capable of expressing the product of the indicator gene. In some embodiments of the method for preparing a recombinant phage, modifying the gene of the parent phage to generate a reproduction-deficient phage is accomplished by HR occurring between the HR plasmid and the genome of the parent phage, and the gene of the parent phage is modified by replacing at least a portion of the parent phage with the indicator gene. In some embodiments, the genetic alteration comprises partial or complete deletion of a gene from the parent phage. Thus, in some embodiments, the method comprises altering the genome of the parent phage such that at least one gene from the parent phage is deleted. In some embodiments, at least 2, 3, 4, or 5 genes are deleted.

[0011] Some embodiments of the method for preparing a recombinant phage can further include generating an engineered strain of the target microorganism. In some embodiments, generating an engineered strain of the target microorganism can include transforming the target microorganism with a plasmid (a "trans plasmid") that encodes and is capable of expressing the altered gene in the recombinant, propagation-deficient phage. Some embodiments of the method for preparing a recombinant phage can further include preparing a homologous recombination plasmid comprising an indicator gene prior to the transforming step. In some embodiments, generating an engineered strain of the target microorganism can include transforming the target microorganism with an HR plasmid comprising the trans plasmid and the indicator gene. In some embodiments of the method for preparing a recombinant phage, the step of altering the genes of a parent phage to generate a propagation-deficient phage is accomplished by infection of an engineered target microorganism with a wild-type parent phage containing both a trans-plasmid and an HR plasmid, such that HR can occur between the genomes of the HR plasmid and the parent phage, and the genes of the parent phage are altered by replacement of at least a portion of the parent phage with an indicator gene, while the plasmid containing the genes to be altered in the propagation-deficient recombinant phage (trans-plasmid) provides the genes in trans to complement the lost or altered genes in the propagation-deficient phage. In a further embodiment of the method for preparing a recombinant phage, the step of deleting genes of a parent phage to generate a propagation-deficient phage is achieved by infection of an engineered target microorganism with a wild-type parent phage containing both a trans-plasmid and an HR plasmid, such that HR can occur between the genomes of the HR plasmid and the parent phage, and the genome of the parent phage is altered by replacement of at least a portion of the parent phage with an indicator gene, while the plasmid containing the gene to be altered in the propagation-deficient recombinant phage (trans-plasmid) provides the gene in trans to complement the lost or altered gene in the propagation-deficient phage.

[0012] In some embodiments of the method for preparing a recombinant phage, the step of altering the genes of a parent phage to generate a propagation-deficient phage is achieved by infection of an engineered target microorganism with a wild-type parent phage that does not contain a plasmid capable of encoding and expressing the gene to be altered in the propagation-deficient phage, but does contain an HR plasmid, such that HR can occur between the genome of the HR plasmid and the parent phage, and the gene of the parent phage is altered by replacement of at least a portion of the parent phage with an indicator gene, while the wild-type parent phage that infects or co-infects the bacterium provides the gene in trans to complement the missing or altered gene in the propagation-deficient phage.

[0013] In some embodiments, isolating specific clones of recombinant phage that are propagation-deficient and capable of expressing the product of the indicator gene can include performing a limiting dilution assay to isolate clones that exhibit expression of the indicator gene. The recombinant phage is derived from a phage specific for E. coli, Salmonella, Listeria, or Staphylococcus. In some embodiments of the method for preparing recombinant phage, the recombinant phage is derived from an Escherichia coli or Salmonella or Listeria or Staphylococcus. In some embodiments of the method for preparing a recombinant phage, the recombinant phage is derived from a phage specific for Salmonella.

[0014] An exemplary embodiment of the present disclosure is a method for detecting a microorganism of interest in a sample, comprising incubating the sample with a recombinant phage according to an embodiment of the present disclosure and detecting a product of an indicator gene, wherein positive detection of the indicator gene product indicates the presence of the microorganism of interest in the sample. In some embodiments of the method for detecting a microorganism of interest in a sample, the sample may be a food sample, an environmental sample, a water sample, or a commercial sample. In some embodiments of the method for detecting a microorganism of interest in a sample, the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single microorganism in the sample. In some embodiments of the method for detecting a microorganism of interest in a sample, the microorganism of interest is Escherichia coli. In some embodiments of the method for detecting a microorganism of interest in a sample, the microorganism of interest is Salmonella.

[0015] Exemplary embodiments of the present disclosure include a kit for detecting a microorganism of interest in a sample, the kit including a recombinant phage according to embodiments of the present disclosure and a substrate for reacting with and detecting the indicator gene product. Exemplary embodiments of the present disclosure also include a system for detecting a microorganism of interest, the system including a recombinant phage according to claim 1 and components for detecting the indicator gene product.

[0016] The present disclosure can be better understood with reference to the following non-limiting drawings. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 illustrates schematically an exemplary method for preparing recombinant, reproductively deficient indicator phage in which the introduction of the reproductively deficient and indicator genes into the parent phage is achieved in a one-step recombination process.

[0018] [Figure 2]FIG. 2 illustrates schematically a "permissive" microorganism transformed with a plasmid expressing genes required for phage propagation and infected with a propagation-deficient indicator phage.

[0019] [Figure 3] Figure 3 illustrates schematically the homologous recombination by co-infection trans-complementation of CBA120 E. coli-specific phage to produce the propagation-deficient indicator phage CBA120.Δgp22.NanoLuc.

[0020] [Figure 4] FIG. 4 is a table illustrating the detection limit of the CBA120.Δgp22.NanoLuc propagation-defective indicator phage in stationary phase E. coli O157:H7 ATCC 43888.

[0021] [Figure 5] FIG. 5 illustrates schematically the propagation of recombinant, propagation-defective indicator phage specific for the E. coli O157:H7 serotype in an engineered E. coli O157:H7 strain transformed with a plasmid expressing the gp22 prohead scaffold protein (a "permissive" E. coli O157:H7 strain).

[0022] [Figure 6] FIG. 6 shows an exemplary growth curve of a propagation-deficient indicator phage, which showed successful growth in the permissive E. coli O157:H7 strain.

[0023] [Figure 7] FIG. 7 outlines the strategy using propagation-defective indicator phages specific for the E. coli O157:H7 serotype.

[0024] [Figure 8]FIG. 8 is a bar graph illustrating the raw signal results of a detection assay performed on log-phase E. coli O157:H7 using a propagation-deficient indicator phage compared to a propagation-competent indicator phage specific for E. coli O157:H7 serotype.

[0025] [Figure 9] FIG. 9 is a bar graph illustrating the signal versus background results of a detection assay using a propagation-deficient indicator phage compared to a propagation-competent indicator phage specific for E. coli O157:H7 serotype, performed on log-phase E. coli O157:H7.

[0026] [Figure 10] FIG. 10 is a bar graph illustrating the raw signal results of a detection assay performed on stationary-phase E. coli O157:H7 using a propagation-deficient indicator phage compared to a propagation-competent indicator phage specific for the E. coli O157:H7 serotype.

[0027] [Figure 11] FIG. 11 is a bar graph illustrating the signal versus background results of a detection assay using a propagation-deficient indicator phage compared to a propagation-competent indicator phage specific for the E. coli O157:H7 serotype, performed on stationary-phase E. coli O157:H7.

[0028] [Figure 12] FIG. 12 is a line graph illustrating the results of specificity determination of propagation-defective indicator phages specific for the E. coli O157:H7 serotype.

[0029] [Figure 13] FIG. 13 illustrates schematically homologous recombination by co-infection trans-complementation of TSP1 Salmonella-specific phage to produce the propagation-defective indicator phage TSP1.Δgp22.NanoLuc.

[0030] [Figure 14] FIG. 14 illustrates schematically the propagation of a Salmonella -specific recombinant propagation-defective indicator phage in an engineered Salmonella strain transformed with a plasmid expressing the gp22 prohead scaffold protein (a "permissive" Salmonella strain).

[0031] [Figure 15] FIG. 15 is a bar graph illustrating the raw signal results of a detection assay using propagation-defective indicator phage in wild-type Salmonella compared to permissive Salmonella.

[0032] [Figure 16] FIG. 16 is a table illustrating the detection limit of SP1.Δgp22.NanoLuc propagation-defective indicator phage in stationary-phase Salmonella typhimurium ATCC 19585.

[0033] [Figure 17] FIG. 17 is a table illustrating the detection limit of SP1.Δgp22.NanoLuc reproductively defective indicator phage in logarithmic phase Salmonella typhimurium ATCC 19585.

[0034] [Figure 18] FIG. 18 illustrates schematically homologous recombination by co-infection trans-complementation of SEA1 Salmonella-specific phage to produce the propagation-defective indicator phage SEA1.Δgp84.NanoLuc.

[0035] [Figure 19] FIG. 19 illustrates schematically the propagation of a Salmonella-specific recombinant propagation-defective indicator phage in an engineered Salmonella strain transformed with a plasmid expressing the gp84 baseplate wedge protein (a "permissive" Salmonella strain).

[0036] [Figure 20] FIG. 20 is a line graph illustrating the raw signal results of a detection assay using SEA1.Δgp84.NanoLuc propagation-defective indicator phage in wild-type Salmonella compared to permissive Salmonella.

[0037] [Figure 21] FIG. 21 is a line graph illustrating the raw signal results of a detection assay using SEA1.Δgp84.NanoLuc propagation-defective indicator phage in wild-type Salmonella strains 7001, 8326, 13076, and 27869.

[0038] [Figure 22] FIG. 22 is a bar graph illustrating a plaque assay of replication of SEA1.Δgp84.NanoLuc propagation-defective indicator phage in wild-type Salmonella strains 7001, 8326, 13076, and 27869.

[0039] [Figure 23] FIG. 23 is a table illustrating the limit of detection of SEA1.Δgp84.NanoLuc reproductively defective indicator phage in logarithmic phase Salmonella Newport ATCC 27869 transformed with AmpR pUC57 SEA1.transgp84.

[0040] [Figure 24] FIG. 24 is a table illustrating the detection limit of the SEA1.Δgp84.NanoLuc propagation-defective indicator phage in stationary-phase Salmonella chloreaesuis ATCC 7001.

[0041] [Figure 25] FIG. 25 is a table illustrating the detection limit of SEA1.Δgp84.NanoLuc reproductively defective indicator phage in logarithmic phase Salmonella chloreaesuis ATCC 7001.

[0042] [Figure 26] Figure 26A is a table illustrating the approximate number of SEA1.NanoLuc replicating phage and SEA1.Δgp84.NanoLuc non-replicating CFU per well. Figure 26B is a table illustrating the RLU signal results of a detection assay using replicating phage and SEA1.Δgp84.NanoLuc compared to a reproduction-deficient indicator phage compared to SEA1.NanoLuc replicating phage specific for Salmonella typhimurium after 2 hours of infection. Figure 26C is a table illustrating the RLU signal results of a detection assay using replicating phage and SEA1.Δgp84.NanoLuc compared to a reproduction-deficient indicator phage compared to SEA1.NanoLuc replicating phage specific for Salmonella typhimurium after 4 hours of infection.

[0043] [Figure 27] FIG. 27 depicts the isolation of recombinant propagation-defective indicator phage using a series of sequential infection and dilution steps to identify propagation-defective indicator phage.

[0044] [Figure 28] FIG. 28 depicts the use of a recombinant, propagation-defective indicator phage encoding a soluble luciferase to detect a microorganism of interest by detection of luciferase, according to an embodiment of the present disclosure.

[0045] [Figure 29] FIG. 29 depicts a filter plate assay for detecting a microorganism of interest using a recombinant reproduction-defective indicator phage according to an embodiment of the present disclosure, whereby the microorganism of interest and the recombinant reproduction-defective indicator phage are incubated in a filter plate and the indicator protein is detected directly without removing the incubation medium.

[0046] [Figure 30]FIG. 30 depicts a "No Concentration Assay" for detecting a microorganism of interest using recombinant propagation-defective indicator phage according to an embodiment of the present disclosure.

[0047] [Figure 31] FIG. 31 depicts a Hybrid Immuno-Phage (HIP) assay for detecting a microorganism of interest using a recombinant reproduction-deficient indicator phage according to one embodiment of the present disclosure, in which an antibody against the microorganism of interest is used to capture the microorganism on the surface of an assay well prior to incubation with a recombinant reproduction-deficient indicator phage. DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description of the Invention definition Unless otherwise specified herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods well known in the art and described in various general and more specific references described throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures used in connection with the laboratory procedures and techniques described herein are those well known and commonly used in the art.

[0049] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0050] As used herein, the terms "a," "an," and "the" can refer to one or more, unless specifically stated otherwise.

[0051] Use of the term "or" is used to mean "and / or," unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0052] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists from sample to sample.

[0053] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface to which a biomolecule can be attached. For example, a solid support can be an assay well (i.e., a microtiter plate or multiwell plate, etc.), or the solid support can be a filter, an array, or a location on a mobile support such as a bead or membrane (e.g., a filter plate or lateral flow strip).

[0054] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction can be non-covalent, e.g., as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently bound) with a solid support.

[0055] As used herein, the terms "reproduction defective" or "reproduction defective" or "replication defective" or "replication defective" refer to an impairment in the ability of a bacteriophage to reproduce. That is, a reproductively defective bacteriophage may be unable to generate new bacteriophage particles due to, for example, the loss of a protein required for capsid assembly. Various deletions, insertions, or substitutions in the bacteriophage genome can render a bacteriophage reproductively defective.

[0056] As used herein, "analyte" refers to a molecule, compound, or cell being measured. An analyte of interest can, in certain embodiments, interact with a binding agent. As described herein, the term "analyte" can refer to a protein or peptide of interest. An analyte can be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes can include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like.

[0057] The terms "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicator moiety" refer to a molecule, or a compound produced by a molecule (such as an enzyme), that can be measured in a quantitative assay. For example, an indicator or indicator moiety can include an enzyme that can be used to convert a substrate to a product that can be measured. An indicator or indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that produces bioluminescent radiation. Alternatively, an indicator or indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used. The term "indicator gene" is used to refer to a gene that encodes an indicator, such as a protein, e.g., an enzyme.

[0058] As used herein, "phage" includes one or more of several viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms. In this disclosure, the term "phage" and related terms include bacteriophages that can invade bacteria and archaea, mycobacteriophages that can invade mycobacteria (a family of bacteria that includes mycobacteria of the Mycobacterium tuberculosis group, which includes the causative agent of tuberculosis, and mycobacterium avis group, which includes the causative agent of tuberculosis), mycophages that can invade fungi, mycoplasma phages, and mycoplasma phages. as well as viruses, such as those capable of infecting protozoa, yeast, and other microscopic living organisms. By large dimension, we mean a size of one millimeter or less. Bacteriophages are viruses that evolved in nature to use bacteria, mycobacteria, or archaea as a means of replicating themselves. In nature, phages attach themselves to microorganisms, inject their DNA (or RNA) into the microorganism, and then induce the microorganism to replicate the phage hundreds or even thousands of times. This is called phage amplification. For example, well-studied phages of Escherichia coli include T1, T2, T3, T4, T5, T7, and lambda; other E. coli phages available in the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. Salmonella phages include TSP1, TSP11, SPN1S, 10, epsilon 15, SEA1, TSP1, and P22. Listeria phages include P100, LMA8, LMA4, LPES1, LipZ5, P40, vB_LmoM_AG20, P70, P100, LP-JS3, LP-ES1, and A511. Staphylococcus phages include staph phage ISP, P4W, virus K, Twort, phi11, 187, P68, and phiWMY.

[0059] As used herein, "late gene region" refers to a region of a viral genome that is transcribed late in the viral life cycle. Late gene regions typically contain the most abundantly expressed genes (e.g., structural proteins assembled into bacteriophage particles). Late genes of bacteriophages are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, from 8 minutes after infection to lysis, class I (e.g., RNA polymerase) is transcribed early, 4-8 minutes later, and class II is transcribed 6-15 minutes later, with overlap in timing between II and III. A late promoter is an active promoter naturally located in such a late gene region.

[0060] As used herein, "enrichment culture" refers to traditional culture, such as incubation in a medium favorable for microbial growth, and should not be confused with other possible uses of the word "enrichment," such as enrichment by 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. Enrichment culture over a period of time can be used in some embodiments of the methods described herein.

[0061] As used herein, "recombination" refers to a genetic (i.e., nucleic acid) modification typically performed in a laboratory to bring together genetic material that would not otherwise be found. The term is used interchangeably herein with the term "modified." As used herein, "RLU" refers to relative light units measured by a luminometer (e.g., GLOMAX® 96) or similar instrument that detects light. For example, detection of the reaction between luciferase and an appropriate substrate (e.g., NANOLUC® by NanoGlo) is often reported in terms of detected RLU.

[0062] Overview Disclosed herein are compositions, methods and systems that exhibit surprising sensitivity for the detection of microorganisms of interest, such as bacteria and archaea, in test samples (e.g., biological, food, water and environmental). Some non-limiting examples of microorganisms of interest include Bacillus spp., Bordetella pertussis, Brucella spp., Campylobacter spp. (such as Campylobacter jejuni), Chlamydia pneumoniae, Cronobacter spp., Clostridium perfringens, Clostridium botulinum, Enterobacter spp., Escherichia spp. (such as Escherichia coli, e.g., E. coli O157:H7 and other Shiga toxin- and enterotoxin-producing strains of Escherichia coli), Klebsiella pneumoniae, Klebsiella oxytoca, Listeria spp. (such as Listeria monocytogenes), Mycoplasma pneumoniae, Pseudomonas spp., Salmonella spp. (e.g., Salmonella typhi, Salmonella The following bacterial species have been identified: Salmonella typhimurium or Salmonella enteritidis, Shigella sonnei, Yersinia spp., Vibrio spp., Staphylococcus spp. (e.g., Staphylococcus aureus), and Streptococcus spp. Detection can be achieved in shorter time frames than previously thought possible using genetically engineered phage in assays performed without enrichment culture, or in some embodiments, with minimal incubation times during which microorganisms can potentially multiply. Equally surprising, potentially high multiplicities of infection (MOIs) or high concentrations of plaque-forming units (PFU) for incubation with test samples have been successfully used. Such high phage concentrations (PFU / mL) were previously said to be detrimental in bacterial detection assays because they were said to cause "lysis from without."However, high concentrations of phage can facilitate finding, binding to, and infecting a small number of target cells.

[0063] The compositions, methods, systems, and kits of the present invention can include recombinant phage for use in detecting a microorganism of interest. In certain embodiments, the present invention can include compositions comprising a recombinant phage having an indicator gene inserted into the late gene region of the phage. Such recombinant phage are referred to as "indicator phage." In certain embodiments, expression of the indicator gene after infection of a host microorganism results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene (i.e., class III) region of the bacteriophage. Recombinant bacteriophages according to embodiments of the present invention can be used to detect viruses such as podoviruses, such as T7 and T7-like; myoviruses, such as T4 and T4-like; siphoviruses, such as T5, P70, and Saka6; and related phages, Cronobacter viruses, such as ViI, ViI-like (or Vi1 viruses according to GenBank / NCBI), Saka2, or Saka4. The phage may be derived from a spp-specific bacteriophage, Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage epsilon 15, Salmonella phage SEA1, 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, Escherichia coli O157:H7 phage CBA120, or another wild-type or engineered bacteriophage.

[0064] Indicator phages according to embodiments of the present invention are reproductively defective, meaning that they are unable to reproduce efficiently or at all after infecting the microorganism of interest being detected. Reproduction-deficient indicator phages according to embodiments of the present invention are made reproductively defective due to the alteration of one or more suitable genes, for example, late genes required for virion assembly. In some embodiments, reproduction-deficient indicator phages according to embodiments of the present invention are made reproductively defective by introducing a mutation into a suitable gene separately from the introduction of the indicator gene. In some other embodiments, reproduction-deficient indicator phages according to embodiments of the present invention are made reproductively defective by replacing at least a portion of a suitable gene with an indicator gene. Reproduction-deficient indicator phages according to embodiments of the present invention can grow or reproduce in a host microorganism engineered to produce the product of the mutated gene required for phage reproduction. Such engineered microorganisms are termed "permissive."

[0065] Reproduction-deficient indicator phages have several advantages over previously described indicator phages. Because reproduction-deficient indicator phages require specially engineered microorganisms for propagation, their potential for production and distribution by inexperienced and / or untrained providers is limited. The production and distribution of damaged and low-quality reagents is a significant problem in the field of diagnostics. By limiting the production and distribution of indicator phages to entities with certain qualifications and meeting certain standards (e.g., through an official certification process), the provision of reproduction-deficient indicator phages reduces the risk of low-quality or damaged indicator phages being produced and distributed to diagnostic operators. Furthermore, because they are unable to propagate in host microorganisms found in the environment, reproduction-deficient indicator phages eliminate the risk that standardized diagnostic reagents containing a defined concentration and / or amount of indicator phages will be contaminated by host microorganisms prior to performing a diagnostic procedure, which could result in an undetected increase in the concentration or amount of indicator phages in the reagent and, consequently, inaccurate detection data. This issue is particularly important in quantitative or semi-quantitative detection, where the concentration or amount of indicator phage used correlates with the intensity of the signal being detected. Furthermore, due to their inability to reproduce in the target microorganism during the diagnostic process, reproduction-deficient indicator phage according to embodiments of the present invention enable more accurate quantitative or semi-quantitative detection of the target microorganism in a sample. The improvement in accuracy results from the ability to control the amount of reproduction-deficient indicator phage found in the sample throughout the detection process. Because new viable indicator phage are not generated during the detection process, only the reproduction-deficient indicator phage initially used can express the indicator gene product after infection. Due to the inherent high expression level of late genes, and because deletion of early genes often does not result in genome replication, reducing the copy number of the indicator gene in each cell, deletion and replacement of late genes ensures high expression of the indicator gene compared to early genes. Reproduction of indicator phage after infection can introduce significant variability into the amount of indicator signal produced during the diagnostic process.Thus, the use of propagation-deficient indicator phages according to embodiments of the present invention allows for easier standardization of quantitative and semi-quantitative detection, improving the accuracy of detection results.

[0066] In some aspects, the present invention includes methods for detecting a microorganism of interest. The methods can use phage for detection of the microorganism of interest. Thus, in certain embodiments, the methods can include detecting a microorganism of interest in a sample by incubating the sample with a recombinant, reproduction-deficient indicator phage that infects the microorganism of interest. In some embodiments, the recombinant, reproduction-deficient indicator phage is a bacteriophage. In certain embodiments, the indicator gene can be inserted into a late gene region of the bacteriophage such that expression of the indicator gene results in production of an indicator gene product after infection of the host microorganism. The methods can include detecting the indicator gene product, wherein positive detection of the indicator gene product indicates the presence of the microorganism of interest in the sample. In some embodiments, the indicator gene product is a protein. In some embodiments, the indicator gene product is a soluble protein.

[0067] In certain embodiments, the present invention can include a system. The system can contain at least a portion of the composition of the present invention. The system can also include at least a portion of the components for performing the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present invention can include a system for rapid detection of a microorganism of interest in a sample, the system including components for incubating the sample with a reproduction-deficient indicator phage specific for the microorganism of interest, where the reproduction-deficient indicator phage includes an indicator gene, and components for detecting the indicator. In yet other embodiments, the present invention includes software for use with the method or system.

[0068] Some embodiments of the present invention solve a need in the field of microbial detection by using a bacteriophage-based method to amplify a detectable signal indicating the presence of bacteria. In certain embodiments, as few as one bacterium is detected. The principles applied herein can be applied to the detection of a variety of microorganisms. Due to the large number of binding sites for phage on the surface of the microorganism and the potential for high-level expression of the encoded indicator, the indicator may be more easily detectable than the microorganism itself. In this way, embodiments of the present invention can achieve enormous signal amplification even from a single infected cell.

[0069] Some embodiments of the present invention disclosed and described herein take advantage of the fact that a single microorganism can bind multiple recombinant reproduction-defective indicator phages according to embodiments of the present invention. After infection by the recombinant reproduction-defective indicator phages, it is detected by an indicator encoded by the recombinant reproduction-defective indicator phages and expressed in the microorganism. This principle allows for the amplification of indicator signals from one or a few cells based on specific recognition of microbial surface receptors. For example, even a single bacterial cell can be exposed to multiple reproduction-defective indicator phages and subsequently express the encoded indicator gene products, amplifying the indicator signal so that the microorganism of interest can be detected with high sensitivity. For example, a single bacterium present in a sample can be detected using embodiments of the present invention. Embodiments of the present invention take advantage of the high specificity of phages capable of binding to specific microorganisms as a way to detect and / or quantify specific microorganisms in a sample. In some embodiments, the present invention takes advantage of the high specificity of reproduction-defective indicator phages.

[0070] Embodiments of the methods and systems of the present invention can be applied to the detection and quantification of various microorganisms (including, but not limited to, bacteria and archaea) in a variety of contexts, including, but not limited to, the detection of pathogens from food, water, and commercial samples. The methods of the present invention provide high detection sensitivity and specificity, as well as rapid detection.

[0071] sample Each of the composition, method, kit, and system embodiments of the present invention allows for rapid detection and / or quantification of a microorganism of interest in a sample. For example, methods according to embodiments of the present invention can be performed in a shortened period of time with excellent results.

[0072] Microorganisms that can be detected in a sample using embodiments of the present invention include, but are not limited to, bacteria that are food or water borne pathogens. Bacteria that can be detected by the present invention include Bacillus spp., Bordetella pertussis, Brucella spp., Campylobacter spp. (such as Campylobacter jejuni), Chlamydia pneumoniae, Cronobacter spp., Clostridium perfringens, Clostridium botulinum, Enterobacter spp., Escherichia spp. (such as Escherichia coli, e.g., E. coli O157:H7 and other Shiga toxin- and enterotoxin-producing strains of Escherichia coli), Klebsiella pneumoniae, Listeria spp. (such as Listeria monocytogenes), Mycoplasma pneumoniae, Salmonella spp. (such as Salmonella typhi, Salmonella typhimurium, or Salmonella enteritidis), Shigella sonnei, Yersinia spp., Vibrio spp., Staphylococcus spp. (e.g., Staphylococcus aureus), and Streptococcus spp.

[0073] The sample may be, but is not limited to, an environmental sample, a food sample, or a water sample. Some embodiments may include a medical or veterinary sample. The sample may be a liquid, solid, or semi-solid. The sample may be a swab of a solid surface. The sample may 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 may be fish, meat such as beef, pork, or lamb, poultry, processed foods, peanut butter, infant formula. Milk (powdered infant formula), powdered milk, tea, starch, eggs, milk, cheese or The sample may be a sample of other dairy products. Medical or veterinary samples include, but are not limited to, blood, sputum, cerebrospinal fluid, fecal samples, and irrigation washes. In some embodiments, irrigation is used to collect a biological sample. Irrigation is the flow of a solution (e.g., saline) over an open wound or an implanted device. Thus, in some embodiments, the biological sample is a wound wash or device wash. In some embodiments, the sample may be a different type of swab.

[0074] In some embodiments, samples can be used directly in the detection methods according to embodiments of the present invention without preparation, concentration, or dilution. For example, liquid samples, including but not limited to milk and juice, can be assayed directly. In other embodiments, samples can be diluted or suspended in a solution, including but not limited to a buffer solution or bacterial culture medium. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. In some embodiments, samples should be maintained within a pH range that promotes recombinant bacteriophage attachment to host bacterial cells. In some embodiments, a preferred pH range may be a range suitable for bacteriophage to attach to bacterial cells. The sample can also be analyzed by Na + , Mg 2+ and K. + It should contain appropriate concentrations of divalent and monovalent cations, including but not limited to:

[0075] In some embodiments, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. During the step in which the bacteriophage attaches to the bacterial cells, the sample can be maintained at a temperature that facilitates bacteriophage activity. Such a temperature is at least about 25°C and not more than about 45°C. In some embodiments, the sample is maintained at about 37°C. In some embodiments, the sample is subjected to gentle mixing or shaking during recombinant bacteriophage binding or infection.

[0076] Embodiments of the present invention can utilize a variety of suitable control samples, for example, a control sample containing no phage or a control sample containing phage without the microorganism of interest can be assayed as a control for background signal levels.

[0077] Reproduction-deficient indicator phage As described in more detail herein, compositions, methods, systems, and kits according to embodiments of the present invention can include a reproductively deficient indicator phage for use in detecting pathogenic microorganisms. In certain embodiments, the present invention includes a recombinant reproductively deficient indicator bacteriophage having a genetic modification(s) that includes an indicator gene and renders the phage reproductively deficient. The above-described genetic modifications can be introduced in a single genetic modification step or in multiple genetic modification steps (such as two or more genetic modification steps). In some embodiments, the present invention can include a composition comprising a reproductively deficient indicator phage.

[0078] The recombinant reproduction-defective indicator phage can include a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene after infection of a host microorganism, such as a bacterium, results in a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene region of the reproduction-defective indicator phage. Late genes encode structural proteins and are therefore generally expressed at higher levels than other phage genes.

[0079] Recombinant, reproduction-deficient indicator phages according to embodiments of the present invention include modifications that render the recombinant phage unable to reproduce after infection of a host organism. Suitable genes and modifications are selected according to a number of considerations. Phage genes suitable for modification are those that affect the phage's ability to reproduce in a host microorganism after infection, but do not affect the ability of the recombinant, reproduction-deficient phage to infect the host microorganism. In some embodiments, the gene to be modified to render the recombinant phage reproduction-deficient is selected so that it is not required for phage genome replication. This ensures that the recombinant phage genome replicates to a typically high copy number, resulting in high copy numbers of the indicator gene. Early and immediate-early genes often fall into the category of genes required for phage genome replication. Early and immediate-early genes (e.g., T7 RNA polymerase) may also be required for expression of genes controlled by late gene promoters, such as indicator genes, in recombinant phages. Thus, immediate-early and early genes, also known as class I or class II genes, may not be suitable for modification. In some embodiments, the gene to be altered to render the recombinant phage propagation-deficient is selected because it is required for mature phage virion production. For example, a gene suitable for alteration or deletion may be a structurally important gene, such as a gene required for virion assembly. In some embodiments, the gene to be altered to render the recombinant phage propagation-deficient is selected to be a late gene required for mature phage virion production but that is not expressed at high copy number. In some embodiments, the propagation-deficient indicator phage can contain two or more altered genes (i.e., one or more).Some examples of genes that may be suitable for modification or deletion to render recombinant phage propagation-deficient include: in bacteriophage T4 and related phages and T4 viruses (e.g., SEA1, Saka4, and TSP12 phages) and the closely related Viu-like viruses (e.g., CBA120, TSP1 phage), some genes that may be suitable for modification are gp4, which encodes the head completion protein; gp20, which encodes the portal vertex protein; gp21, which encodes the prohead core scaffolding protein and protease; gp22, which encodes the prohead scaffolding protein; gp25, which encodes the baseplate wedge subunit; gp26, which encodes the baseplate hub subunit; gp53, which encodes the baseplate wedge component; and gp54, which encodes the baseplate-tail tube initiator. In podoviruses (T7, MP87 phages), some genes that may be suitable for modification are gp4, which encodes the head completion protein; gp20, which encodes the portal vertex protein; gp21, which encodes the prohead core scaffolding protein and protease; gp22, which encodes the prohead scaffolding protein; gp25, which encodes the baseplate wedge subunit; gp26, which encodes the baseplate hub subunit; gp53, which encodes the baseplate wedge component; and gp54, which encodes the baseplate-tail tube initiator. In T. elegans, some of the genes that may be suitable for modification are gp6.7, which encodes the virion protein; gp7.3, which encodes the tail protein; gp8, which encodes the head-to-tail connector protein; gp9, which encodes the scaffolding protein; and gp13. In Siphovirus (T5, a P70-related phage), some of the genes that may be suitable for modification are Gp150, which encodes the prohead protease, and gp152, which encodes the portal protein. It should be understood that the above list is non-limiting and that other genes may be modified in various phages.

[0080] In some embodiments, a reproductively deficient indicator phage according to embodiments of the present invention comprises a mutation in a suitable gene. Such a mutation may be an amber mutation, an ochre mutation, a base substitution, a deletion or insertion, or a combination of any of the above types of mutations. The mutation or combination thereof may render the gene selected for modification dysfunctional by altering the encoded protein structure, suppressing transcription or expression of the modified gene (e.g., by altering the promoter), causing premature termination of transcription or expression, etc. In some other embodiments, in a reproductively deficient indicator phage, the suitable gene is modified by replacing at least a portion of the suitable gene with an indicator gene. As a result, the recombinant phage becomes reproductively deficient and incorporates the indicator gene sequence. In some embodiments, replacing at least a portion of the suitable gene in the phage with an indicator gene may be preferable, rather than introducing one or more mutations into the suitable gene, to avoid reversion or suppression of one or more mutations in the suitable gene and restoring the recombinant phage to reproductive competence.

[0081] In some embodiments, the reproductively defective indicator bacteriophage is a podovirus such as T7, T7-like, a myovirus such as T4, T4-like, a ViI, ViI-like (or Vi1 virus according to GenBank / NCBI), a Cronobacter such as Saka2 or Saka4. spp-specific bacteriophage, Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage epsilon 15, Salmonella phage SEA1, Salmonella phage Spn1s, Salmonella phage P22, Salmonella phage TSP1, Salmonella phage TSP11, Listeria phage LipZ5, Listeria phage P40, Listeria phage vB_LmoM_AG20, Listeria phage P70, Listeria phage A511, Listeria phage LMA4, Listeria phage LMA8, Listeria phage LPES1, Listeria phage LPJP1, Staphylococcus phage P4W, Staphylococcus phage K, Staphylococcus phage Twort, Staphylococcus phage SA97, Staphylococcus phage ISP, Escherichia coli It can be derived from the O157:H7 phage CBA120, or another wild-type or engineered bacteriophage.In some embodiments, the indicator bacteriophage is a podovirus such as T7, T7-like, myovirus such as T4, T4-like, ViI, ViI-like (or Vi1 virus according to GenBank / NCBI), Cronobacter spp-specific bacteriophage such as Saka2 or Saka4, Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage epsilon 15, Salmonella phage SEA1, 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, Escherichia coli The reproductively deficient indicator phage is derived from a bacteriophage having a genome at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the genome of O157:H7 phage CBA120, or another wild-type or engineered bacteriophage. In some embodiments, the reproductively deficient indicator phage is derived from a phage that is highly specific to a particular microorganism. For example, the reproductively deficient indicator bacteriophage can be prepared from a bacteriophage derived from an environment specific to bacteria found in a particular environment.

[0082] The selection of an indicator gene to be inserted into a propagation-defective indicator phage can be guided by various considerations. For example, most phages are capable of packaging DNA several percent larger than their native genome. Given this consideration, a smaller indicator gene may be a more appropriate choice for engineering bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (approximately 500-600 bp to encode), while FLuc is approximately 62 kDa (approximately 1,700 bp to encode). For comparison, the T7 genome is around 40 kbp, while the T4 genome is approximately 170 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacterium (i.e., not be part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. NANOLUC® by PROMEGA® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC® in combination with NanoGlo (also by PROMEGA®), an imidazopyrazinone substrate (furimazine), can provide a robust signal with low background. In some embodiments, two or more indicator genes can be inserted into the propagation-deficient phage. For example, two or more copies (e.g., two copies) of the same indicator gene can be inserted, which can improve the signal strength and / or signal-to-noise ratio of assays using propagation-deficient indicator phage. In another example, different indicator genes, such as two different indicator genes, can be inserted, which can enable bimodal signal detection. For example, the NANOLUC® gene can be inserted together with a gene encoding green fluorescent protein (GFP), or the NANOLUC® gene can be inserted together with a gene encoding a different luciferase, such as firefly luciferase.

[0083] Indicator genes can encode various biomolecules or may themselves be detectable biomolecules. For example, indicator genes can encode detectable polypeptides or proteins. In another example, indicator genes can be genes that express a detectable product or an enzyme that produces a detectable product. In yet another example, indicator genes can encode or include a detectable nucleic acid. For example, indicator genes can encode a detectable aptamer, such as RNA Mango, or can contain a nucleic acid sequence that is detectable by real-time polymerase chain reaction (RT-PCR). In some embodiments, the product of the indicator gene can be a detectable enzyme. The indicator gene product can produce light and / or be detectable by a 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 function as indicator moieties. For example, in some embodiments, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. The luciferase can be one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®. Other engineered luciferases or other enzymes that generate a detectable signal can also be suitable indicator moieties.

[0084] Genetic modifications to a reproduction-deficient indicator bacteriophage can include the insertion, deletion, or replacement of a small fragment of nucleic acid, a substantial portion of a gene, or an entire gene. In some embodiments, the inserted or replaced nucleic acid comprises a non-native sequence. For example, a non-native indicator gene can be inserted into the bacteriophage genome so that it is under the control of a bacteriophage promoter. The non-native indicator gene can be inserted to replace at least a portion of the sequence of a late phage gene, and the insertion of the indicator gene renders the resulting recombinant phage reproduction-deficient. Including stop codons in all three reading frames of the indicator gene can help increase expression by reducing read-through, also known as leaky expression. This strategy can also eliminate the possibility of fusion proteins being produced at low levels, which would manifest as background signal that cannot be separated from the phage. Thus, in some embodiments, the non-native 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 contain a phage polypeptide. In some embodiments, the present invention includes a genetically modified, reproductively defective indicator bacteriophage containing a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels like the viral capsid protein, but also does not shut down like similar endogenous bacterial or early bacteriophage genes. In some embodiments, the late promoter is a T4, T7, or ViI-like promoter, or another phage promoter similar to those found in wild-type phages.

[0085] In some embodiments, expression of the indicator gene of the propagation-defective indicator phage in the microorganism of interest following infection with the propagation-defective indicator phage results in the production of a soluble protein product. In some embodiments, the non-native indicator gene is not adjacent to a gene encoding a structural phage protein and therefore does not result in a fusion protein. Unlike systems that use a fusion of a detection moiety to a capsid protein (a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., a soluble luciferase). In some embodiments, the indicator or reporter ideally does not include the phage structure. That is, the indicator or reporter is not attached to the phage structure. Thus, the gene for the indicator or reporter is not fused to other genes in the genome of the propagation-defective indicator phage. This significantly increases the sensitivity of detection assays in which propagation-defective indicator phage according to embodiments of the present invention are used (sensitivity can be increased up to the detection of a single microorganism in a sample) and simplifies the assay, allowing the assay to be completed in two hours or less for some embodiments, as opposed to the several hours required by additional purification steps required by constructs that produce detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding constraints that may alter the conformation of the enzyme active site or access to the substrate. For example, less than 2 hours may be sufficient for the assay at a concentration of 10 bacterial cells per mL of sample.

[0086] Furthermore, fusion proteins, by definition, limit the number of moieties attached to a protein subunit in a bacteriophage. For example, use of a commercially available system designed to serve as a platform for fusion proteins would result in approximately 415 copies of the fusion moiety, corresponding to approximately 415 copies of gene 10B capsid protein in each T7 bacteriophage particle. Without this constraint, infected bacteria could be expected to express many more copies of the detection moiety (e.g., luciferase) than can fit on the bacteriophage.

[0087] In some embodiments of recombinant propagation-deficient indicator phage, a late promoter (such as a class III promoter, e.g., from T7, T4, ViI, or Saka) is used to transcribe the indicator gene. Such later promoters have high affinity for the RNA polymerase of the same phage, which transcribes the genes for the structural proteins assembled into phage particles. Because each phage particle contains dozens or hundreds of copies of these molecules, such proteins are the most abundant proteins produced by phage. The use of a viral late promoter can ensure optimally high-level expression of the indicator gene product, such as luciferase. The use of a late viral promoter derived from, specific for, or active under the original wild-type bacteriophage from which the propagation-deficient indicator phage is derived (e.g., the T4, T7, ViI, or Saka late promoter in T4-, T7-, ViI-, or Saka-based systems) can further ensure optimal expression of the detection moiety. The use of standard bacterial (non-viral / non-bacteriophage) promoters can be detrimental to expression in some cases, as these promoters are often down-regulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production). Thus, in some embodiments, propagation-deficient indicator phages are engineered to encode and express high levels of a soluble (free) indicator moiety, preferably using a location in the genome that does not limit expression to the number of subunits of the phage structural components.

[0088] In some embodiments, reproductively deficient indicator phages are designed to optimize desired traits for use in assays for the detection of a microorganism of interest. In some embodiments, bioinformatics and previous analysis of genetic modifications 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 species of bacteria. In other embodiments, the gene encoding the phage tail protein can be optimized to recognize and bind to an entire genus of bacteria or a specific group of species within a genus. In this way, the phage can be optimized to detect a broader or narrower group of pathogens. In some embodiments, the reproductively deficient indicator phage can be designed to improve reporter gene expression. Additionally and / or alternatively, in some instances, the reproductively deficient indicator phage can be designed to increase the burst size of the phage to improve detection. Engineering a reproductively deficient indicator phage to produce an increased copy number of the phage genome after infection or to increase the expression level of a late gene will result in an increased burst size.

[0089] In some embodiments, the stability of the propagation-deficient indicator phage can be optimized to improve shelf life. For example, enzybiotic solubility can be increased to increase subsequent phage stability. Additionally and / or alternatively, the thermal stability of the propagation-deficient indicator phage can be optimized. Thermostable phage better preserve functional activity during storage, thereby increasing shelf life. Thus, in some embodiments, thermal stability and / or pH tolerance can be optimized.

[0090] The compositions of the present invention can comprise one or more reproductively deficient indicator bacteriophages and one or more indicator genes. In some embodiments, the compositions can comprise a cocktail of different reproductively deficient indicator phages specific for different microorganisms of interest. Such cocktails can be used for the simultaneous detection of multiple microorganisms of interest. In some embodiments, the compositions can comprise a cocktail of different reproductively deficient indicator phages capable of encoding and expressing the same or different indicator proteins. In some embodiments, the cocktail of reproductively deficient bacteriophages comprises at least two different types of reproductively deficient indicator bacteriophages.

[0091] Method for preparing (producing) reproduction-deficient indicator bacteriophages Methods for generating propagation-deficient indicator phages according to embodiments of the present invention can begin with the selection of parent phages for genetic modification. For example, some bacteriophages are highly specific for target microorganisms, including specificity for specific strains or serotypes of target microorganisms. This presents an opportunity for highly specific detection. The parent phages can be wild-type phages found in any environment or engineered phages. Methods according to embodiments of the present invention utilize the high specificity of binding associated with bacteriophages that recognize and bind to specific microorganisms of interest as a means of amplifying the signal and thereby detecting low levels of microorganisms (in some cases, down to a single microorganism) present in a sample. For example, bacteriophages specifically recognize surface receptors of specific microorganisms and thus specifically infect such microorganisms. Therefore, bacteriophages are suitable for targeting microorganisms of interest. Some embodiments of the present invention utilize the specificity of binding and the high-level genetic expression capabilities of indicator bacteriophages for rapid and sensitive targeting to infect and facilitate detection of target microorganisms of interest. Thus, some embodiments of methods for preparing recombinant propagation-defective indicator phage can include steps involving selecting parent phage that specifically infect a target microorganism of interest.

[0092] Some embodiments of the method for preparing a recombinant, propagation-defective indicator phage include one or more steps of modifying the genes of a parent phage to generate a recombinant, propagation-defective phage. For example, some embodiments may include one or more steps of introducing one or more mutations into a suitable gene to render the parent phage propagation-defective. Such suitable genes and mutations are described elsewhere in this document.

[0093] Some embodiments of methods for preparing recombinant reproduction-defective indicator phage include modifying a gene in a parent phage to generate a recombinant reproduction-defective phage. To reproduce, the reproduction-defective phage requires an engineered strain of the phage's host microorganism (such as a bacterium) capable of expressing the product of the modified gene to render the phage reproduction-defective. Such an engineered strain can be termed "permissive." Thus, some embodiments of methods for preparing recombinant reproduction-defective indicator phage can include generating such a permissive engineered strain of the host microorganism. Some embodiments of methods for preparing recombinant reproduction-defective indicator phage can include infecting a permissive engineered strain of the host microorganism with the reproduction-defective indicator phage. Some embodiments of methods for preparing recombinant reproduction-defective indicator phage can include preparing a homologous recombination plasmid / vector containing an indicator gene. Some embodiments of methods for preparing recombinant reproduction-defective indicator phage can include transforming the homologous recombination plasmid / vector into a permissive engineered host microorganism infected with the reproduction-defective indicator phage. Some other embodiments of the method for preparing a recombinant, reproductively defective indicator phage can include transforming a homologous recombination plasmid / vector into a permissive engineered host microorganism and then infecting the transformed, permissive engineered host microorganism with the reproductively defective indicator phage. In some embodiments, the infection of the permissive engineered host microorganism and the transformation of the homologous recombination plasmid / vector into the permissive engineered host microorganism can be accomplished in the same step. In other embodiments, the infection of the permissive engineered host microorganism and the transformation of the homologous recombination plasmid / vector into the permissive engineered host microorganism are accomplished in two or more steps. When the permissive engineered host microorganism accepts the reproductively defective phage and the homologous recombination plasmid / vector, homologous recombination occurs between the plasmid / vector and the phage genome. Recombinant, reproductively defective phage (reproduction-defective indicator phage) containing the indicator gene can then be isolated.

[0094] In some embodiments, the gene of the parent phage that is altered to render the phage reproductively defective can be altered by replacing at least a portion of the parent phage with an indicator gene. Thus, some embodiments of methods for preparing a recombinant reproductively defective indicator phage include preparing a homologous recombination plasmid / vector containing an indicator gene flanked by sequences of genes targeted for deletion in the parent phage to render it reproductively defective. The homologous recombination plasmid / vector is then transformed into a permissive engineered host microorganism infected with the parent phage, thereby allowing homologous recombination to occur between the plasmid / vector and the parent phage genome. Some other embodiments of methods for preparing a recombinant reproductively defective indicator phage include transforming the homologous recombination plasmid / vector into a permissive engineered host microorganism, followed by infecting the transformed permissive engineered host organism with the reproductively defective indicator phage, thereby allowing homologous recombination to occur between the plasmid / vector and the parent phage genome. In some embodiments, infection of a permissive engineered host microorganism and transformation of the homologous recombination plasmid / vector into the permissive engineered host organism are accomplished in the same step, allowing homologous recombination to occur between the plasmid / vector and the parent phage genome. Recombinant recombination-deficient phages (propagation-deficient indicator phages) containing the indicator gene can then be isolated.

[0095] In some embodiments of the method for preparing recombinant propagation-deficient indicator phage, the introduction of the propagation-deficient and indicator genes into the parent phage is accomplished in a one-step recombination process. The recombination strategy for such a process is illustrated in FIG. 1. An advantage of such an embodiment is the simplification of the process for generating propagation-deficient indicator phage. Another advantage of such an embodiment is that a reporter gene can be used for both detection and isolation of the propagation-deficient indicator phage. If a genetic alteration is introduced to confer propagation deficiency in a propagation-competent phage containing an indicator gene (propagation-competent indicator phage), both phage with and without the propagation-deficient alteration will grow, making screening for indicator phage more difficult.

[0096] FIG. 3 illustrates the phase transition between a homologous recombination (HR) plasmid and a parent phage genome in a host microorganism, resulting in the generation of an indicator phage according to one embodiment of the present invention. The recombination process is illustrated schematically. In the described homologous recombination process, the phage is a CBA120 E. coli phage, and the reporter gene is a NANOLUC® reporter gene. Thus, the host microorganism is E. coli in the described embodiment. It should be understood that FIG. 3 is intended to be exemplary and non-limiting, and that other phages, corresponding host organisms, and reporter genes can be used. In some embodiments, it may be preferable to utilize phages isolated from the environment for the production of reproductively defective indicator phages. In this way, reproductively defective indicator phages specific to naturally occurring microorganisms can be generated.

[0097] Various methods are known for designing and preparing homologous recombination plasmids.Various methods are known for transforming bacteria with plasmids, including heat shock, F-pilus-mediated bacterial conjugation, electroporation, and other methods.Various methods are also known for isolating specific clones after homologous recombination.Some methods of the embodiments described herein utilize specific strategies.

[0098] Some embodiments of methods for preparing a reproductively defective indicator bacteriophage include selecting a parent phage that specifically infects a target microorganism of interest; determining the native sequence in the late region of the genome of the selected parent phage; annotating the genome and identifying suitable late genes of the selected parent phage, where modification of the suitable late genes is intended to render the parent phage reproductively defective; designing sequences for homologous recombination adjacent to the major late gene and including a codon-optimized reporter gene; incorporating the designed sequences for homologous recombination into a plasmid / vector; transforming the plasmid / vector into a target microorganism containing a plasmid encoding a functional suitable late gene; selecting for the transformed target microorganism; infecting the transformed microorganism with the selected parent 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. Enrichment of the propagation-defective recombinant phage fraction over the parental phage fraction can be performed in whole or in part in permissive target cells containing the trans-plasmid. Some embodiments include further repeating the limiting dilution and titer steps after the first limiting dilution assay, as needed, until the recombinant phage represent a detectable fraction of the mixture. For example, in some embodiments, the limiting dilution and titering 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. A 1:30 recombinant:parental ratio is expected in some embodiments to yield an average of 3.2 transducing units (TU) per 96 plaques (e.g., in a 96-well plate). The initial ratio of recombinant to parental phage can be determined by performing a limiting dilution assay based on TCID50 (tissue culture infectious dose 50%), as previously described in U.S. Patent Application No. 15 / 409,258. By Poisson distribution, a 1:30 ratio generates a 96% probability of observing at least 1 TU in any of the 96 wells.

[0099] Some embodiments include the design (and, if necessary, preparation) of sequences for homologous recombination necessary for the insertion of an indicator gene. In some embodiments, the homologous recombination sequences are designed to replace the late gene to render the parent phage reproductively defective. In some embodiments, the indicator gene sequence comprises a codon-optimized reporter gene preceded by an untranslated region. The untranslated region can include a phage late gene promoter and a ribosome entry site. In some embodiments, the inserted genetic construct further comprises its own exogenous, dedicated promoter to drive expression of the indicator gene. The exogenous promoter is included in addition to any endogenous promoters in the phage genome. Because phage produce polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron in the transcript. Conventional recombinant constructs use only endogenous phage promoters to drive inserted genes. 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.

[0100] Numerous methods and commercial products are known for preparing plasmids. For example, plasmids can be prepared using a combination of PCR, site-directed mutagenesis, restriction enzyme digestion, ligation, cloning, and other techniques. Synthetic plasmids can also be commercially ordered (e.g., GeneWiz). Cosmids can also be used, or the CRISPR / CAS9 system can be used to selectively edit the bacteriophage genome. Some embodiments of methods for preparing indicator bacteriophages include designing a plasmid that can be easily recombined with a starting bacteriophage genome to generate a recombinant genome. In designing the plasmid, some embodiments include adding a codon-optimized reporter gene, such as a luciferase gene. Some embodiments further include adding elements to an upstream untranslated region. For example, an upstream untranslated region can be added before the sequence encoding the start codon of the NANOLUC® reporter gene. The untranslated region can include a promoter, such as a T4, T4-like, T7, T7-like, ViI, or ViI-like promoter. The untranslated region may also contain a ribosome entry / binding site (RBS), also known as a "Shine-Dalgarno sequence" in bacterial systems. Either or both of these elements, or other untranslated elements, can be embedded within a short upstream untranslated region made of randomized sequences with approximately the same GC content as the rest of the phage genome. The randomized region should not contain an ATG sequence, as this will act as the start codon.

[0101] As described elsewhere in this document, isolation and propagation of recombinant propagation-defective indicator phages according to embodiments of the present invention can be performed only in "permissive" host microorganisms that express the modified gene(s) that render the propagation-defective indicator phages propagation-defective. Such "permissive" microorganisms, e.g., bacteria, can be engineered by transforming them with a plasmid expressing a gene required for phage propagation. Figure 2 illustrates cells of such a "permissive" microorganism transformed with a plasmid expressing a gene required for phage propagation and infected with a propagation-defective indicator phage. The plasmid containing the gene required for phage propagation is selected to be compatible with the homologous recombination plasmid used to insert the indicator gene into the phage genome. For example, the plasmid expressing the gene required for phage propagation and the homologous recombination plasmid can be selected to contain different antibiotic resistance markers so that both plasmids can be simultaneously maintained in the host organism. In another example, the plasmid expressing the gene required for phage propagation and the homologous recombination plasmid are selected to contain compatible origins of replication so that they do not interfere with each other. Examples of compatible plasmids are pUC-derived plasmids using the ori origin of replication and pBAV1k-T5-GFP plasmid using the RCR (rolling circle replication) origin of replication. Because propagation-defective phages require an engineered "permissive" strain of host microorganism to propagate, some embodiments of methods for preparing recombinant propagation-defective indicator phages include generating an engineered strain of the target microorganism capable of expressing the product of the gene to be altered in the propagation-defective phage to make it propagation-defective. In some embodiments, generating an engineered strain of the target microorganism involves transforming the target microorganism with a plasmid encoding and capable of expressing the gene(s) to be altered in the recombinant propagation-defective phage. Alternatively, the required gene can be integrated into the target microorganism genome by a variety of other methods, such as by transposon, homologous recombination, site-specific recombination / integration, or others.

[0102] Figure 27 outlines an example process for isolating propagation-deficient recombinant phage from a mixture of parental and propagation-deficient indicator phage resulting from homologous recombination. In the first step (402), a permissive host microorganism transformed with a homologous recombination plasmid and a plasmid expressing the desired phage genes is infected with the parental phage, resulting in progeny phage (434) with a mixture of parental and propagation-deficient recombinant indicator phage at a very low ratio of parental to propagation-deficient indicator phage. The resulting phage mix is ​​diluted (404) and plated into 96-well plates (406) to yield an average of 5 recombinant transducing units (TU) per plate (9.3 PFU / well). The 96-well plates are then assayed for reporter gene activity, as described below, to identify wells containing propagation-deficient indicator phage (436) compared to wells containing parental phage (440). A permissive host microorganism (438) containing a plasmid expressing the desired phage gene is added to each well (408); for example, if the host microorganism is a bacterium, each well can contain approximately 50 μL of turbid bacterial culture. This allows the propagation-defective indicator phage to replicate and produce a soluble reporter gene product (442). After an incubation step (410) (e.g., 5 hours at 37° C.), the wells can be screened for the presence of the reporter gene product (442). Any positive well may have been inoculated with a single propagation-defective indicator phage; at this stage, the mixture can contain a ratio of approximately 10 parent phage:1 recombinant, an enrichment over the original ratio. If necessary (e.g., if the recombinant:total ratio is lower than 1:30), the progeny of this enrichment culture (412) can be subjected to additional limiting dilution assay(s) (414) to increase the ratio and determine the actual concentration of recombinant, propagation-deficient indicator phage transducing units.For example, if the ratio was 1:384 recombinant:PFU (as determined by plaque assay performed in permissive bacteria), approximately 5 recombinant TU could be aliquoted (414) from the previous positive well along with 1920 contaminating total phage (5 x 384 = 1920) per 96-well plate (416), resulting in approximately 20 mostly parental phage seeded per well of the second dilution assay plate (420) (1920 PFU / 96 well = 20 PFU / well). Any positive luciferase well could have been seeded with a single recombinant, propagation-deficient indicator phage along with 19 parental phage. These wells could then be analyzed for the presence of luciferase (442).

[0103] After addition of the host microorganism and incubation (418), the soluble reporter gene product and phage are present at approximately 20:1 (420). This ratio can be verified by TU50 titration for recombinants, a limiting dilution assay based on a tissue culture infectious dose 50 (TCID50) assay that scores for reporter gene product activity instead of cell killing, and a plaque assay for total PFU. Finally, a plaque assay (422) can be performed to screen for recombinants expressing the reporter gene product (446). A small number of individual (e.g., n = 48) plaques can be individually picked and screened for luciferase activity in a third multiwell plate (426) (436). In some embodiments, this approach should ensure that enough plaques are screened, so that approximately 3 indicator bacteriophages are in the mix of plaques being screened, based on the known ratio of recombinants to total phage. A single plaque can be picked from the plate and placed into each well of a 96-well plate (424), and a reporter gene product assay (426) can be performed to determine which wells contained phage exhibiting reporter gene product activity (442). Wells exhibiting such activity (428) represent pure recombinant, propagation-defective indicator phage (434), while wells lacking such activity (430) represent pure parental phage (432). Individual plaques can then be suspended in buffer (e.g., 100 μL of buffer) or medium, and aliquots (e.g., about 5 μL) can be added to wells containing host microbial cultures and assayed after incubation (e.g., about 45 minutes to 1 hour at 37°C). Positive wells are expected to contain pure cultures of propagation-defective indicator phage. Certain embodiments can include additional rounds of plaque purification. Thus, as illustrated by Figure 27, propagation-deficient indicator phages generated by homologous recombination of a plasmid designed for recombination with a parent phage genome can be isolated from a mixture containing a very low percentage (e.g., 0.005%) of indicator bacteriophages.

[0104] Following isolation, large-scale production can be performed to obtain high-titer propagation-deficient indicator phage stocks suitable for use in detection methods according to embodiments of the present invention. Production and preparation of propagation-deficient indicator phage stocks can include purification of the propagation-deficient indicator bacteriophage from any free detection moieties produced during production of the propagation-deficient indicator bacteriophage in bacterial culture. Standard phage purification techniques, such as sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size-exclusion chromatography, and dialysis or derivative techniques (e.g., Amicon brand concentrators - Millipore, Inc.), can be used to purify some embodiments of the phages of the present invention. As a result of the purification procedure, the propagation-deficient indicator phage stocks can be substantially free of any reporter product gene generated during production. Removal of residual indicator gene products present in the propagation-deficient indicator phage stocks can substantially reduce the background signal observed when the propagation-deficient indicator phage is used to detect a microorganism of interest in a sample.

[0105] Method for using propagation-defective indicator phages to detect microorganisms. As mentioned herein, in certain embodiments, the present invention can include a method of using a reproduction-defective indicator phage to detect a microorganism. The method of using a reproduction-defective indicator phage to detect a microorganism according to embodiments of the present invention can be embodied in a variety of ways.

[0106] In one embodiment, the invention includes a method for detecting a microorganism of interest in a sample, the method comprising the steps of incubating the sample with a reproduction-defective indicator phage that infects the microorganism of interest, the reproduction-defective indicator phage comprising an indicator gene such that expression of the indicator gene following infection by the microorganism of interest results in production of the indicator gene product, and detecting the indicator gene product, wherein positive detection (i.e., detection of the presence, amount, or level of the indicator gene product) indicates that the microorganism of interest is present in the sample. In yet another embodiment, the invention includes a method for detecting a microorganism of interest in a sample, the method comprising the steps of incubating the sample with a reproduction-defective indicator phage that infects the microorganism of interest, the reproduction-defective indicator phage comprising an indicator gene such that expression of the indicator gene following infection by the microorganism of interest results in production of a soluble indicator gene product, and detecting the soluble indicator gene product, wherein positive detection (i.e., detection of the presence, amount, or level of the soluble indicator gene product) indicates that the microorganism of interest is present in the sample. In yet another embodiment, the present invention can include a method for detecting a microorganism of interest in a sample, the method comprising the steps of incubating the sample with a reproduction-defective indicator phage that infects the microorganism of interest, the reproduction-defective indicator phage comprising an indicator gene such that expression of the indicator gene following infection by the microorganism of interest results in production of a soluble indicator gene product, and detecting the soluble indicator gene product, wherein a positive detection (i.e., detection of the presence, amount, or level of soluble indicator gene product protein) indicates that the microorganism of interest is present in the sample. In a variation of the above embodiment, the microorganism of interest can be a bacterium of interest.For example, in an exemplary embodiment, the invention includes a method for detecting a bacterium of interest in a sample, the method comprising the steps of incubating the sample with a reproduction-defective indicator phage that infects the bacterium of interest, the reproduction-defective indicator phage comprising an indicator gene such that expression of the indicator gene following infection by the bacterium of interest results in production of a soluble indicator gene product, and detecting the indicator gene product, wherein positive detection of the indicator gene product indicates that the bacterium of interest is present in the sample.

[0107] In certain embodiments, methods using reproductively deficient indicator phages to detect a microorganism of interest (such methods may be referred to as "assays") may be performed utilizing a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments of the present invention using reproductively deficient indicator bacteriophages (i.e., indicator bacteriophages) may be performed in the following order depending on the sample type, sample size, and assay format: 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, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36 This assay can allow for rapid detection of specific bacterial strains with total assay times of less than 0.0, 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. For example, the amount of time required can be slightly shorter or longer depending on the strain of bacteriophage and the strain of bacteria to be detected in the assay, the type and size of the sample to be tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants.

[0108] Figure 28 illustrates a strategy for using a propagation-deficient indicator phage that produces soluble luciferase according to an embodiment of the present invention. In this method, the propagation-deficient indicator phage can be engineered to express soluble luciferase. Expression of the luciferase is driven by a viral capsid promoter (e.g., a bacteriophage T7 or T4 late promoter), resulting in high expression. In the embodiment illustrated in Figure 28, at least a portion of a sample (500) containing the microorganism (502) to be detected is placed in a spin column filter, centrifuged to remove excess liquid, and an appropriate multiplicity of propagation-deficient indicator phage (504) engineered to express soluble luciferase (503) is added. The infected cells can be incubated for a time sufficient for infection to occur (e.g., 30-240 minutes at 37°C). In some embodiments, cell lysis may occur. In other embodiments, the cells may not lyse. The propagation-defective indicator phage (504) and free luciferase (503) in the lysate can then be collected, for example, by centrifugation, and the level of luciferase in the filtrate can be quantified using a luminometer (518). Alternatively, a high-throughput method can be used, whereby samples are added to a 96-well filter plate and assayed for luciferase directly in the original 96-well filter plate without a final centrifugation step, after all procedures listed above. Alternatively, other simplified or self-contained formats can be used, as previously described. Such methods may not require centrifugation or other separation of any components after infection with the propagation-defective indicator phage. In some embodiments, a single device with two, three, four, or more compartments can be used to perform the infection and incubation steps of the assay, followed by detection with an appropriate device, for example, luminescence detection with a handheld luminometer.

[0109] Figure 29 depicts a filter plate assay for detecting a microorganism of interest using a reproduction-deficient indicator phage according to an embodiment of the present invention. Briefly, a sample (616) containing the microorganism of interest (618) is added to a well (602) of a multi-well filter plate (604) and spun (606) to concentrate the sample by removing liquid from the sample. Reproduction-deficient indicator phage (620) is added to the well and incubated (608) with additional medium sufficient for adsorption and a time period sufficient for the reproduction-deficient indicator phage to infect the target microorganism of interest and advance the phage life cycle (610) (e.g., approximately 45 minutes to 2 hours) to achieve late gene production, which typically occurs late in the infection cycle (but without the production of any mature virus particles by the reproduction-deficient indicator phage). Finally, a luciferase substrate is added to react with any luciferase present (624). The resulting radiation is measured in a luminometer (614) that detects luciferase activity (626).

[0110] In certain embodiments, the assay can be performed without concentrating the microorganism of interest at or near the capture surface. Figure 30 illustrates a "no-concentration assay" for detecting a microorganism of interest using a reproduction-defective indicator phage according to an embodiment of the present invention. Aliquots of reproduction-defective indicator phage (714) are dispensed into individual wells (702) of a multiwell plate (704), followed by the addition of a test sample aliquot containing the microorganism of interest (712) and incubation (706) (e.g., 45 minutes at 37°C) for a period sufficient for the phage to produce a soluble indicator (716) (e.g., luciferase). The plate wells (708) containing the soluble indicator and reproduction-defective indicator phage can then be assayed (710) to measure indicator activity in the plate (718) (e.g., luciferase assay). In this embodiment, the test sample is not concentrated (e.g., by centrifugation) but is simply incubated directly with the reproduction-defective indicator phage for a period of time and then assayed for luciferase activity.

[0111] In some embodiments, samples can be enriched prior to testing by incubation under conditions that promote growth, 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 type and size of the sample.

[0112] In some embodiments, the reproduction-defective indicator phage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by the amplified signal generated by the indicator moiety. Thus, the method can include detecting the indicator moiety produced upon reproduction-defective indicator phage infection, where detection of the indicator indicates the presence of a microorganism of interest, such as a bacterium of interest, in the sample. In an exemplary embodiment, the invention can include a method for detecting a bacterium of interest in a sample, comprising incubating the sample with a reproduction-defective indicator phage that infects the bacterium of interest, where the reproduction-defective indicator phage comprises an indicator gene inserted into a late gene region of the phage such that expression of the indicator gene following infection of the bacterium of interest results in production of a soluble indicator gene product, and detecting the indicator gene product, where positive detection (i.e., detection of the presence, level, or amount) of the indicator gene product indicates the presence of the bacterium of interest in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of the bacterium of interest present in the sample.

[0113] As described in more detail herein, methods and systems according to embodiments of the present invention can utilize a range of concentrations of reproduction-deficient indicator phage to infect target microorganisms (e.g., bacteria) that may be present in a sample. In some embodiments, the reproduction-deficient indicator phage is added to a sample at a concentration sufficient to rapidly find, bind to, and infect target microorganisms (e.g., bacteria) that are present in the sample in very low numbers, such as single cells. In some embodiments, the concentration of reproduction-deficient indicator phage may be sufficient to find, bind, and infect target bacteria in less than one hour. In other embodiments, these events may occur in less than two or three hours after addition of the reproduction-deficient indicator phage to the sample. For example, in certain embodiments, the reproduction-deficient indicator phage concentration for the incubation step is 1×10 5 > PFU / mL, 1×10 6 > PFU / mL, 1×10 7PFU / mL or greater than 1 × 10 8 More than PFU / mL.

[0114] In some embodiments of the method for detecting a microorganism of interest in a sample, prior to incubating the sample with a reproduction-deficient indicator phage that infects the microorganism of interest, the reproduction-deficient indicator phage can be purified to be free of any residual indicator protein that may be produced during production of the infectious agent stock. Thus, in certain embodiments, the method can include purifying the reproduction-deficient indicator phage. The recombinant reproduction-deficient indicator phage can be purified by various methods, for example, by using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. Purification can have the added benefit of removing phage that do not carry DNA (i.e., empty phage or "ghosts").

[0115] In some embodiments of the methods of the present invention, microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or several target microorganisms can be added directly to an assay vessel, such as a spin column, microtiter well, or filter, and the assay is performed in the assay vessel. Various embodiments of such assays are disclosed herein.

[0116] In many embodiments of the method, the assay is carried out using a multi-well plate. For example, an aliquot of the test sample can be directly dispensed into the wells of a multi-well plate, and the propagation-defective indicator phage can be added to the wells. After a period sufficient for infection, a lysis buffer can be added together with a substrate for the indicator moiety (e.g., a luciferase substrate for a luciferase indicator) and assayed for the detection of the indicator signal. Some embodiments of the method can be carried out in a filter plate. Some embodiments of the method can be carried out with or without concentrating the sample before infection with the propagation-defective indicator phage.

[0117] The choice of plate (or any other container in which detection can occur) can affect the detection step. For example, some plates can contain a colored or white background, which can affect the detection of light emission. Generally speaking, white plates have higher sensitivity but also result in higher background signals. Plates of other colors can produce lower background signals but also have slightly lower sensitivity. Furthermore, one reason for background signals is light leakage from one well to another adjacent well. Some plates have white wells, while the remaining plates are black. This allows for high signals within the wells but prevents light leakage between wells, thereby reducing background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal of the assay.

[0118] Methods according to embodiments of the invention can include various other steps to increase sensitivity. For example, as described in more detail herein, methods can include steps for washing the captured, infected microorganisms (such as bacteria) after adding the propagation-deficient indicator phage but before incubation to remove excess propagation-deficient indicator phage and / or luciferase or other reporter proteins that may contaminate the propagation-deficient indicator phage preparation.

[0119] Methods according to embodiments of the present invention may include one or more steps related to sample preparation, which may be referred to as "sampling" or "sampling step." In some embodiments, samples can be used directly in methods according to embodiments of the present invention without preparation, concentration, or dilution. For example, liquid samples can be assayed directly. In other embodiments, samples can be diluted or suspended in a solution, including, but not limited to, a buffer solution or bacterial culture medium. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. In some embodiments, the sample should be maintained within a pH range that promotes the attachment of the propagation-deficient indicator phage to the target microorganism, such as the target bacterium. In some embodiments, a preferred pH range may be suitable for the propagation-deficient indicator phage to attach to bacterial cells. The sample should also contain appropriate concentrations of divalent and monovalent cations, including, but not limited to, Na, Mg, and K.

[0120] Preferably, throughout the detection assay, the sample is maintained at a temperature that maintains the viability of any target microorganisms potentially present in the sample. During the step in which the propagation-deficient indicator phage attaches to bacterial cells, the sample is preferably maintained at a temperature that facilitates the activity of the propagation-deficient indicator phage. Such a temperature is at least about 25°C and not more than about 45°C. In some embodiments, the sample is maintained at about 37°C. In some embodiments, the sample is subjected to gentle mixing or shaking during binding or attachment of the propagation-deficient indicator phage to the target microorganism.

[0121] Sampling can be performed using a variety of methods. In some embodiments, the sample (e.g., a food sample) is first liquefied, and a solid support, e.g., a solid support or beads, is immersed in the liquid sample. In some embodiments, the solid support is first immersed in culture medium in a tube before sampling. In some embodiments, the solid support is dried before sampling. In some embodiments, the liquid sample is first cultured for an enrichment period of less than 24 hours, less than 12 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours ("culture enrichment"). In other embodiments, the sample can be enriched after capture of the microorganism of interest on the solid support. In some embodiments, the solid support with the microorganisms can be incubated in growth medium to increase the number of microorganisms. This step is referred to as "incubation enrichment." In such embodiments, the enrichment period can be 1, 2, 3, 4, 5, 6, 7, or up to 8 hours or longer, depending on the type and size of the sample.

[0122] In some embodiments, the detection of the target microorganism can be completed without culturing the sample as a way to increase the population of the microorganism. For example, in certain embodiments, the total time required for detection is 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, less than 45 minutes, or less than 30 minutes. Minimizing the time to obtain results is important in various applications, such as food and environmental testing for pathogens.

[0123] Methods according to embodiments of the present invention can include a step intended to infect a microorganism of interest with a reproduction-deficient indicator phage. For example, the reproduction-deficient indicator phage is contacted or can be contacted with the microorganism of interest by known methods, some of which are described herein. Upon contact with the microorganism of interest, the reproduction-deficient indicator phage infects the microorganism of interest and expresses the indicator gene. The infection time, i.e., the period between when the sample is first contacted with the reproduction-deficient indicator phage and when the detection step begins (e.g., when a substrate for the enzyme indicator moiety is added to the sample contacted with the reproduction-deficient indicator phage), can vary depending on the type of reproduction-deficient indicator phage and the concentration of the microorganism of interest in the sample. The use of a device in which the microorganism of interest, such as bacteria, is captured on a solid support can significantly reduce the time required for infection; for example, the infection time can be one hour or less, whereas in standard assays in which a solid support is not used to capture bacteria, infection typically takes at least four hours. In certain embodiments, the infection time for the methods disclosed herein is less than 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, hi some embodiments, the infection time is about 1 hour, about 2 hours, or about 3 hours.

[0124] The method according to an embodiment of the present invention may further include a step related to detecting the signal produced by the indicator. The indicator produced by expression of the indicator gene can be detected using known methods. For example, one or more signal-producing components can be reacted with the indicator to generate a detectable signal. In some embodiments, the indicator may be a bioluminescent compound. If the indicator is an enzyme, amplification of the detectable signal can be achieved by reacting the enzyme with one or more substrates or an additional enzyme and substrate to produce a detectable reaction product. In another signal-producing system, the indicator may be a fluorescent compound, in which case manipulation of the indicator enzyme is not required to produce a detectable signal. For example, fluorescent molecules including fluorescein and rhodamine and their derivatives and analogs are suitable for use as indicators in such systems. In yet another embodiment, the indicator moiety may be a cofactor, and amplification of the detectable signal can be achieved by reacting the cofactor with an enzyme and one or more substrates or an additional enzyme and substrate to produce a detectable reaction product. In some embodiments, the detectable signal is colorimetric. While the selection of a particular indicator is not critical to the present invention, it should be noted that the indicator may be capable of generating a detectable signal by itself, or may be detectable by an instrument, or may be detectable in conjunction with one or more additional signal-generating components, such as an enzyme / substrate signal-generating system. In some embodiments, the detection step will require the addition of a substrate for the indicator enzyme to function. The substrate can be added in a variety of ways. In some embodiments, the reaction between the indicator (e.g., luciferase) and the substrate can continue for 30 minutes or more, with detection at various time points being desirable to optimize sensitivity. In some embodiments, luminometer readings can be taken initially and at 3, 5, 10, or 15 minute intervals until the reaction is complete.

[0125] Some embodiments of the methods of the present invention include one or more steps related to detecting the indicator signal, which can be referred to as "detection." Detecting the product indicator gene can include detecting its enzymatic activity. Detecting the product of the indicator gene can include detecting light emission or optical density. In some embodiments, the compartment of the device or container where the substrate is mixed with the test sample is transparent so that any optical signal resulting from infection and subsequent incubation with the substrate can be detected without the need to remove the sample from the compartment of the device or container. In this case, the signal can be detected through the wall of the compartment of the device or container. In some embodiments, the device or container containing the reacted sample is inserted into an instrument for detecting the resulting signal. In other embodiments, a detection instrument is used to scan the device containing the reacted sample.

[0126] In some embodiments, a luminometer can be used to detect the reaction between the indicator (e.g., luciferase) and the substrate. Detection of RLUs can be achieved by a luminometer, or other machines or devices can be used; some examples are the GLOMAX® 20 / 20 and GLOMAX® from PROMEGA® (Madison, WI). In some embodiments, a spectrophotometer, CCD camera, or CMOS camera can detect color changes and other light emissions. While absolute RLUs are important for detection, a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is also required to reliably detect single cells or small numbers of cells. The background signal can be obtained by measuring a control sample containing no microorganisms using the same procedure described above. In some embodiments, detection of a signal from a reporter or indicator gene can include the use of instruments that employ, for example, photodiode or PMT (photomultiplier tube) technology. In some embodiments, a handheld luminometer can be used to detect the signal. Suitable PMT handheld luminometers are available from 3M (Maplewood, MN), BIOCONTROL® (Seattle, WA), and CHARM SCIENCES® (Lawrence, MA). Suitable photodiode handheld luminometers are available from HYGIENA® (Camarillo, CA) and NEOGEN® (Lansing, MI). While these handheld luminometers typically produce much lower readings compared to traditional luminometers (such as GLOMAX® or GLOMAX® 20 / 20) for the same sample, multiple experiments have shown that the signal produced was sufficient to be detected by these handheld luminometers. The ability to use these handheld devices to detect microorganisms also offers convenience and flexibility that are often lacking in detection methods using traditional, non-handheld detection devices.

[0127] In some embodiments, the reproduction-defective indicator phage is genetically engineered to contain a gene for an enzyme, such as luciferase, that is produced only upon infection of the microorganism that the phage specifically recognizes and infects. In some embodiments, the indicator moiety is expressed late in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number. Thus, in some embodiments utilizing reproduction-defective indicator phage, the invention includes a method for detecting a microorganism of interest, comprising capturing at least one sample microorganism of interest, incubating the at least one microorganism of interest with a plurality of reproduction-defective indicator phage, allowing time for infection and expression of the soluble indicator moiety, and detecting the indicator moiety, wherein detection of the indicator moiety demonstrates the presence of the microorganism of interest in the sample.

[0128] For example, in some embodiments, the test sample microorganisms of interest can be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a 0.45 μm pore size spin filter or plate filter). In some embodiments, the propagation-defective indicator phage is added in a minimal volume to the sample captured directly on the filter. In some embodiments, the microorganisms captured on the filter or plate surface are then washed one or more times to remove excess unbound propagation-defective indicator phage. In some embodiments, the culture medium (e.g., Luria-Bertani broth, also referred to herein as LB, buffered peptone water, also referred to herein as BPW, or Tryptic soy broth or tryptone soy broth, also referred to herein as TSB, can be added for an additional incubation period to allow for a sufficiently high level of expression of the gene encoding the indicator moiety. In some embodiments, the incubation step with the propagation-deficient indicator phage need only be long enough to achieve a sufficient level of expression of the gene encoding the indicator moiety to allow a specified level of detectable signal from the indicator moiety (e.g., about 100-10,000 RLU / sec or about 200-5,000 RLU / sec) or a specified level of signal-to-noise ratio (e.g., 1-500, 5-200, or 10-100).

[0129] In some embodiments, an aliquot of a test sample containing a microorganism of interest can be added to a spin column, and after infection with a propagation-deficient indicator phage and washing as necessary to remove any excess propagation-deficient indicator phage, the amount of soluble indicator detected will be proportional to the amount of propagation-deficient indicator phage in the infected microorganism of interest.

[0130] A soluble indicator (e.g., luciferase) released into the surrounding liquid upon bacterial lysis can then be measured and quantified. In certain embodiments, the solution is spun through a filter, and the filtrate is collected for assay in a new receptacle (e.g., in a luminometer) after addition of a substrate for the indicator enzyme (e.g., luciferase substrate). Alternatively, the indicator signal can be measured directly in the filter. Thus, in an exemplary embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with the portion of the sample that remains on the filter or that is bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a regular 96-well plate), and the substrate reaction can be detected by placing the plate directly in a luminometer. For example, in one embodiment, the present invention includes a method for detecting a target microorganism of interest, comprising the steps of infecting cells of the target microorganism captured on a 96-well filter plate with a plurality of propagation-deficient indicator phages capable of expressing luciferase after infection; washing away excess propagation-deficient indicator phages; adding LB broth and allowing time (e.g., 30-120 minutes, 60-120 minutes, or 80-100 minutes, e.g., about 90 minutes) for the propagation-deficient indicator phages to express luciferase and lyse the target microorganism; and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity indicates the presence of the target bacterium in the sample.

[0131] In another embodiment, the present invention includes a method for detecting a target microorganism of interest, comprising the steps of infecting cells in a liquid solution or suspension in a 96-well plate with a plurality of propagation-deficient indicator phages capable of expressing luciferase after infection; allowing time (e.g., 30-120 minutes; 60-120 minutes, or 80-100 minutes, e.g., about 90 minutes) for the propagation-deficient indicator phages to express luciferase and lyse the target microorganism; and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity indicates the presence of the target microorganism 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 vegetable wash. In some embodiments, the liquid solution or suspension can be a consumable test sample, such as vegetable wash. The liquid solution or suspension may be concentrated LB broth, trypsin / tryptone soy broth, peptone water, or vegetable wash fortified with nutrient broth. In some embodiments, the liquid solution or suspension may be bacteria diluted in LB broth.

[0132] In some embodiments, lysis of the microorganism of interest can occur before, during, or after the detection step. Infected, unlysed cells may, in some embodiments, be detectable after addition of luciferase substrate. Without complete cell lysis, luciferase can exit the cell and / or luciferase substrate can enter the cell. Thus, for embodiments utilizing a spin filter system, where only luciferase released into the lysate is analyzed in the luminometer (not luciferase still inside intact bacteria), lysis is required for detection. However, for embodiments utilizing filter plates or 96-well plates with samples in solution or suspension, where the original plate filled with intact and lysed cells is assayed directly in the luminometer, lysis is not required for detection.

[0133] In some embodiments, the reaction between the indicator moiety (e.g., luciferase) and the substrate can continue for 30 minutes or more, and detection at various time points can be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as the solid support and luciferase as the indicator, luminometer readings can be taken initially and at 10 or 15 minute intervals until the reaction is complete.

[0134] Surprisingly, the high concentrations of reproductively deficient indicator phage utilized to infect the test sample successfully achieve detection of extremely low numbers of target microorganisms in a very short time frame. Incubation of the test sample with the phage, in some embodiments, only needs to be long enough for a single phage life cycle. In some embodiments, the reproductively deficient indicator phage concentration for this incubation step is 7×10 6 , 8×10 6 , 9×10 6 , 1.0×10 7 , 1.1×10 7 , 1.2 × 10 7 , 1.3 × 10 7 , 1.4×10 7 , 1.5×10 7 , 1.6×10 7 , 1.7×10 7 , 1.8×10 7 , 1.9×10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 or 1.0 × 10 8 More than PFU / mL.

[0135] Embodiments of the methods of the present invention are capable of detecting individual microorganisms. Thus, in certain embodiments, the methods are capable of detecting ≦10 cells of a microorganism (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9 microorganisms) present in a sample. For example, in certain embodiments, the propagation-deficient indicator phage is highly specific for a bacterium of interest. In certain embodiments, the propagation-deficient indicator phage can distinguish a bacterium of interest in the presence of other types of bacteria. In certain embodiments, the propagation-deficient indicator phage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the propagation-deficient indicator phage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample.

[0136] Many phages used for infection have previously been associated with "lysis from without," which kills target cells and prevents the generation of a useful signal. Cleanup of prepared stocks of propagation-deficient indicator phages, as described herein (e.g., cleanup by cesium chloride isopycnic density gradient ultracentrifugation), can help alleviate this problem. In addition to removing any contaminating luciferase associated with propagation-deficient indicator phages, this cleanup can also remove ghost particles (particles that have lost their DNA). Ghost particles can lyse bacterial cells through "lysis from without," causing premature cell death and thereby preventing the generation of an indicator signal. Electron microscopy demonstrates that crude phage lysates (i.e., before cesium chloride cleanup) can contain greater than 50% ghost particles. These ghost particles can contribute to the premature death of microorganisms due to the action of many phage particles puncturing the cell membrane. Thus, ghost particles may have contributed to previous problems where high PFU concentrations were reported to be harmful. Furthermore, purified preparations of propagation-defective indicator phage allow the assay to be performed without a wash step, thereby allowing the assay to be performed without an initial enrichment step. However, it should be understood that some embodiments of the methods of the present invention include an initial enrichment step, and in some embodiments, this enrichment step allows for a shorter enrichment incubation time.

[0137] Some embodiments of the method of the present invention can further include a confirmatory assay. Various assays for confirming initial results, usually at later time points, are known in the art. For example, samples can be cultured as described in the Examples (e.g., CHROMAGAR®, DYNABEADS® assay), PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm the initial results.

[0138] In certain embodiments, the methods of the invention combine detection by infectious agent with the use of binding agents (e.g., antibodies) to purify and / or concentrate a microorganism of interest, such as a bacterium of interest, from a sample. For example, in certain embodiments, the invention provides a method for detecting a microorganism of interest in a sample, comprising capturing the microorganism from the sample on a prior support using a capture antibody specific for the microorganism of interest, such as a bacterium of interest; In one embodiment, the method comprises the steps of: incubating a sample with a reproduction-defective indicator phage that infects a target bacterium, wherein the reproduction-defective indicator phage comprises an indicator gene inserted into a late gene region of the reproduction-defective indicator phage such that expression of the indicator gene after infection of the bacterium of interest results in a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample.

[0139] For example, Figure 31 depicts a hybrid immunophage (HIP) assay for detecting a microorganism of interest using a reproduction-defective indicator phage according to an embodiment of the present invention. Samples are first added to microtiter plate wells coated with a microorganism-specific antibody (802). The plate is then washed to facilitate binding of the microorganism of interest to the capture antibody (804). After a sufficient time to allow complete capture, a solution containing a microorganism-specific reproduction-defective indicator phage is added to each sample (806). Incubation with the phage results in binding and attachment of single or multiple phages to the captured microorganism (808). Finally, the sample is incubated to facilitate luciferase expression, which results in cell lysis and release of soluble luciferase (810).

[0140] Systems and Kits In some embodiments, the present invention includes systems (e.g., automated systems or kits) comprising components for carrying out the methods disclosed herein. In some embodiments, a propagation-deficient indicator phage is included in a system or kit of the present invention. The methods described herein may also utilize such propagation-deficient indicator phage systems and / or kits. Some embodiments described herein are particularly suitable for automation and / or kits given the minimal amount of reagents and materials required to carry out the methods. In certain embodiments, each of the components of the kit may comprise a self-contained unit deliverable from a first site to a second site.

[0141] In some embodiments, the present invention includes a system or kit for rapid detection of a microorganism of interest in a sample. The system or kit, in certain embodiments, can include a component for incubating a sample with a reproduction-defective indicator phage specific for the microorganism of interest, where the reproduction-defective indicator phage includes an indicator moiety, and a component for detecting the indicator moiety. In some embodiments of both the systems and kits of the present invention, the reproduction-defective indicator phage is capable of specifically infecting the bacterium of interest and includes an indicator gene inserted into the late gene region of the reproduction-defective indicator phage as the indicator moiety, such that expression of the indicator gene upon infection of the microorganism results in a soluble indicator protein product. Some systems further include a component for capturing the microorganism of interest on a solid support. In certain embodiments, the system or kit can include a device comprising a solid support, the solid support including a cell-binding component and a signal-detection component, the signal-detection component being capable of detecting an indicator gene product produced from infection of the microorganism in the sample with the reproduction-defective indicator phage. In some embodiments, the signal-detection component is a luminometer, which can be a handheld device.

[0142] In other embodiments, the present invention includes a method, system, or kit for rapid detection of a microorganism of interest in a sample, the method, system, or kit including a reproduction-deficient indicator phage component specific for the microorganism of interest, where the reproduction-deficient indicator phage comprises an indicator moiety, and a component for detecting the indicator moiety. In certain embodiments, the reproduction-deficient indicator phage is highly specific for a particular microorganism, such as bacteria. In some embodiments, the reproduction-deficient indicator phage can distinguish a microorganism of interest, such as bacteria, in the presence of other types of microorganisms. In certain embodiments, the system or kit detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific microorganisms of interest in a sample.

[0143] In certain embodiments, the present invention can include a system or kit for rapid detection of a microorganism of interest in a sample, the system or kit including a device having a first compartment containing a propagation-deficient indicator phage. The device can further include a second compartment containing a substrate and / or a third compartment containing a medium. One or more of these compartments are sealed and separated from the rest of the device by a snap seal, and failure of the snap seal causes the contents of the compartment to exit the compartment and mix with the sample. Alternatively, the system or kit can further include separate containers containing the substrate and / or medium.

[0144] In certain embodiments, the systems and / or kits can further include components for washing the captured microbial sample. Additionally or alternatively, the systems and / or kits can further include components for determining the amount of indicator moiety, where the amount of detected indicator moiety corresponds to the amount of microorganisms in the sample. For example, in certain embodiments, the systems or kits can include a luminometer or other device for measuring luciferase enzyme activity.

[0145] In some embodiments, the system and / or kit may include components for isolating a microorganism of interest from other components in a sample. In some systems and / or kits, the same components may be used for multiple steps. In some systems and / or kits, the steps are automated or controlled by a user via computer input, and / or a liquid-handling robot performs at least one step. In computerized systems, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination thereof).

[0146] Thus, in certain embodiments, the present invention can include a system or kit for rapid detection of a microorganism of interest in a sample, the system or kit including: a component for incubating the sample with a reproduction-deficient indicator phage specific for the microorganism of interest, the reproduction-deficient indicator phage comprising an indicator moiety; a component for capturing the microorganism of interest from the sample on a solid support; a component for washing the captured microorganism of interest to remove unbound reproduction-deficient indicator phage; and a component for detecting the indicator moiety. In some embodiments, the same component can be used for the capturing, incubating, and / or washing steps (e.g., a filter component). Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, the amount of the indicator moiety detected corresponding to the amount of the microorganism in the sample. Such a system can include various embodiments and subembodiments similar to those described above for the rapid detection method of a microorganism. In certain embodiments, the microorganism is a bacterium. In a computerized system, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination thereof). In some embodiments, the system may include components for isolating a microorganism of interest from other components in a sample.

[0147] In certain embodiments, the present disclosure includes a system or kit including components for detecting a microorganism of interest, including components for isolating at least one microorganism from other components in a sample, components for infecting at least one microorganism with a plurality of reproduction-deficient indicator phages, components for lysing at least one infected microorganism to release reproduction-deficient indicator phages present in the microorganism, and components for detecting the reproduction-deficient indicator phages, or perhaps with greater sensitivity, soluble proteins encoded and expressed by the reproduction-deficient indicator phages, wherein detection of the reproduction-deficient indicator phages or soluble protein products of the reproduction-deficient indicator phages indicates the presence of the microorganism in the sample. The reproduction-deficient indicator phages may be NANOLUC® reproduction-deficient indicator phages carrying the NANOLUC® indicator gene.

[0148] In other embodiments, the present disclosure may include a kit for rapid detection of a microorganism of interest in a sample, the kit including a component for incubating the sample with a reproduction-deficient indicator phage specific for the microorganism of interest, where the reproduction-deficient indicator phage includes an indicator moiety; a component for capturing the microorganism of interest from the sample on a solid support; a component for washing the captured microorganism of interest to remove unbound reproduction-deficient indicator phage; and a component for detecting the indicator moiety. In some embodiments, the same component can be used for the capturing, incubating, and / or washing steps. Some embodiments also include a component for determining the amount of the microorganism of interest in the sample, where the amount of the indicator moiety detected corresponds to the amount of the microorganism of interest in the sample. Such a kit may include various embodiments and subembodiments similar to those described above for the rapid detection method of a microorganism. In certain embodiments, the microorganism is a bacterium. In some embodiments, the kit may include a component for isolating the microorganism of interest from other components in the sample.

[0149] These 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 carrying out the recited method. The components need not be integrally connected or positioned relative to one another in any particular way. The present disclosure encompasses any suitable arrangement of components relative to one another. For example, the components need not be in the same room. However, in some embodiments, the components are connected to one another in an integral unit. In some embodiments, the same component can perform multiple functions.

[0150] Computer System and Computer-Readable Medium In certain embodiments, the present disclosure can include a system. The system can include at least a portion of the composition of the present disclosure. The system can also include at least a portion of the components for performing the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present disclosure can include a system for rapid detection of a microorganism of interest in a sample. The system can include at least a portion of the composition of the present disclosure. The system can also include at least a portion of the components for performing the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present disclosure can include a system for rapid detection of a microorganism of interest in a sample, the system including the apparatus described above. For example, the apparatus can include a first compartment containing a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, the construct including a promoter and an indicator gene, and the solid support including a cell-binding component. In some embodiments, the system also includes a handheld detection device.

[0151] The systems described in the present technique, or any of its components, may be embodied in the form of a computer system, typical examples of which include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps comprising the methods of the present technique.

[0152] The computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include a memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include a storage device. The storage device may be a hard disk drive or a removable storage drive, such as a floppy disk drive or an optical disk drive. The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit connects the computer to other databases and the Internet via an I / O interface. The communication unit enables the transfer of data to other databases as well as the reception of data from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that can connect the computer system to databases and networks such as LANs, MANs, WANs, and the Internet. Thus, the computer system may facilitate input from a user via an input device accessible to the system via the I / O interface.

[0153] A computing device typically includes 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.) that stores instructions that, when executed by the server's processor, cause the computing device to perform its intended functions. Suitable implementations of operating systems and the general functionality of computing devices are known or commercially available and are readily implemented by those skilled in the art, especially in light of the disclosure herein.

[0154] A computer system executes a set of instructions stored in one or more storage elements to process input data. The storage elements may also hold data or other information as desired. The storage elements may be in the form of information sources or physical memory elements present in a processing machine.

[0155] The environment can include various data storage and other memory and storage media, as described above. These can reside in a variety of locations, such as on storage media local to (and / or residing within) one or more of the computers, or remote from any or all of the computers via a network. In a particular set of embodiments, information can reside on a storage area network ("SAN"), familiar to those skilled in the art. Similarly, any files necessary to perform functions ascribed to a computer, server, or other network device can be stored locally and / or remotely, as appropriate. When the system includes computing devices, each such device can include hardware elements that can be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices, such as random access memory (“RAM”) or read-only memory (“ROM”), disk drives, optical storage devices, and solid-state storage devices, as well as removable media devices, memory cards, flash cards, and the like.

[0156] Such devices may also include computer-readable storage medium readers, communication devices (e.g., modems, network cards (wireless or wired), infrared communication devices, etc.), and working memory, as described above. The computer-readable storage medium reader may be connected to or configured to receive computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The systems and various devices will also typically include numerous software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs, such as a client application or web browser. It should be appreciated that alternative embodiments can have numerous variations of what is described above. For example, customized hardware may be used, and / or particular elements may be implemented in hardware, software (including portable software, such as applets), or both. Additionally, connections to other computing devices, such as network input / output devices, may be used.

[0157] 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 storage and / or transmission of information, such as computer-readable instructions, data structures, program modules or other data, including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by a system device. Based on the disclosure and teachings provided herein, one of ordinary skill in the art will recognize other ways and / or methods to implement the various embodiments.

[0158] Computer-readable media may include, but are not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, content addressable memory ("CAM"), DDR, flash memory such as NAND flash or NOR flash, ASICs, configured processors, optical storage, magnetic tape or other magnetic storage, or any other medium from which a computer processor can read instructions. In one embodiment, a computing device may include a single type of computer-readable medium, such as random access memory (RAM). In other embodiments, a computing device may include two or more types of computer-readable media, such as random access memory (RAM), a disk drive, and a cache. A computing device may communicate with one or more external computer-readable media, such as an external hard disk drive or an external DVD or Blu-ray drive.

[0159] As described above, embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include, for example, processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, including C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In some embodiments, a computing device includes a single processor. In other embodiments, a device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronic devices, such as PLCs, programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electrically programmable read-only memories (EPROMs or EEPROMs), or other similar devices.

[0160] The computing device includes a network interface. In some embodiments, the network interface is configured for communication via a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface may enable communication over a network such as a CDMA, GSM, UMTS, or other cellular communication network. In some embodiments, the network interface may enable a 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 a suitable computing device may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include data storage in addition to or in place of the network interface.

[0161] Some embodiments of a suitable computing device may include or communicate with numerous external or internal devices, such as a mouse, a CD-ROM, a DVD, a keyboard, a display, audio speakers, one or more microphones, or any other input or output device. For example, the computing device may communicate with a variety of user interface devices and displays. The displays may use any suitable technology, including, but not limited to, LCD, LED, CRT, etc.

[0162] A set of instructions for execution by a computer system may include various commands that instruct a processing machine to perform specific tasks, such as steps that make up the methods of the present technique. The set of instructions may be in the form of a software program. Further, the software may be in the form of a collection of separate programs, a program module with a larger program, or portions of a program module, as found in the present technique. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be in response to user commands, results of previous processing, or a request made by another processing machine.

[0163] While the present disclosure has been disclosed with reference to certain specific embodiments, numerous modifications, variations, and variations to the described embodiments are possible without departing from the scope and spirit of the disclosure as defined in the appended claims. Accordingly, it is intended that the present disclosure not be limited to the described embodiments, but rather that its full scope be defined by the following claims and equivalent language. [Example]

[0164] The following examples describe the detection of small numbers of cells, even single bacteria, with reduced time to results and are intended to illustrate, not limit, the present disclosure.

[0165] Example 1: Creation and isolation of propagation-defective indicator phages from bacteriophages specific for E. coli O157:H7 serotype Using homologous recombination as illustrated in Figure 3, a propagation-deficient indicator phage specific for the E. coli O157:H7 serotype was constructed from a parent phage specific for the E. coli O157:H7 serotype. To generate the propagation-deficient indicator phage, the coding sequence for the gp22 prohead scaffold protein of the parent phage was replaced with the NANOLUC® coding sequence. E. coli O157:H7 was transformed with both a homologous recombination plasmid containing the NANOLUC® gene flanked by matching bacterial genomic sequences flanking gp22 (HR plasmid in Figure 3) and a plasmid containing an expression cassette for gp22 (pBAV.gp22 in Figure 3), each under separate antibiotic selection conditions, to ensure that the transformed bacteria contained both plasmids. These doubly transformed bacteria were infected with the parent phage to allow homologous recombination with the HR plasmid, deleting the phage copy of gp22, which is then provided in trans by the gp22-encoding plasmid. Following homologous recombination, a series of titering and enrichment steps were used to isolate specific recombinant bacteriophages expressing NANOLUC®. Large-scale production was performed to obtain high-titer stocks of propagation-defective indicator phage suitable for use in detection assays. As illustrated in Figure 5, the propagation-defective indicator phage (designated CBA120Δgp22 NanoLuc) was unable to grow in wild-type E. coli O157:H7 and was therefore propagated in an engineered E. coli O157:H7 strain transformed with a high-copy pUC-based plasmid expressing the gp22 prohead scaffold protein (a "permissive" E. coli O157:H7 strain). As illustrated by the growth curves shown in Figure 6, the propagation-defective indicator phage was able to grow in the permissive E. coli O157:H7 strain. Growth was successful in the O157:H7 strain. Cesium chloride isopycnic density gradient centrifugation was used to separate the phage particles from the contaminating luciferase protein to reduce background.

[0166] Example 2: Testing of Reproduction-Defective Indicator Phages Specific for E. coli O157:H7 Serotype in a Detection Assay The strategy using the E. coli O157:H7 serotype-specific propagation-deficient indicator phage CBA120Δgp22 NanoLuc is shown in Figure 7. After infection with E. coli O157:H7, the propagation-deficient indicator phage produced soluble luciferase. The propagation-deficient indicator phage was unable to form phage heads due to the missing gp22 protein. The propagation-deficient indicator phage did not produce viable daughter phage.

[0167] To evaluate the activity of the CBA120Δgp22 NanoLuc propagation-deficient indicator phage, its detection activity was compared with that of CBA120 NanoLuc, a propagation-competent indicator phage specific for E. coli O157:H7, which contains the NANOLUC® gene inserted after the major capsid protein gene, gp23, under the control of a T4 late gene promoter. Logarithmic and stationary-phase cultures of E. coli O157:H7 (ATCC 43888) were diluted to obtain the approximate number of CFUs indicated on the x-axis of Figures 8-11 when a 100 μl sample was used. Each sample was infected with either CBA120 NanoLuc or CBA120Δgp22 NanoLuc for 2 hours at 37°C. Lysis buffer and luciferase substrate were added, and the samples were read in a luminometer. Five replicate measurements were performed at each CFU level for each phage. The RLU values ​​for each CFU were averaged. The mean value for each CFU level reading was used and divided by the mean of the 0 CFU readings to calculate the signal / background values ​​plotted on the y-axis in Figures 8-11.

[0168] The above experiments were performed on E. coli O157:H7 culture samples in logarithmic phase, where bacterial cells typically produce higher signal levels due to high levels of transcription and protein expression. The results are illustrated in Figures 8 and 9. The above experiments were also performed on E. coli O157:H7 culture samples in stationary phase, where bacterial cells typically produce lower signal levels due to lower levels of transcription and protein expression. The results are illustrated in Figures 10 and 11. In Figures 8 and 9, open bars indicate results obtained with the propagation-defective indicator phage (labeled CBA12.Δgp22.NL), and solid bars indicate results for the positive control (labeled CBA120NL). Figures 8-11 show that the propagation-defective indicator phage performed comparable to the positive control.

[0169] Example 3: Examination of the specificity of propagation-defective indicator phages specific to E. coli O157:H7 serotype The specificity of the propagation-defective indicator phage specific for the E. coli O157:H7 serotype was examined. Detection assays were performed as described in the previous example to detect a range of bacteria. The results are illustrated in Figure 12. A luciferase signal was produced upon detection of E. coli O157:H7 or engineered permissive E. coli O157:H7. No luciferase signal was detected upon attempted detection of non-target bacteria, including several E. coli serotypes.

[0170] Example 4: Creation and isolation of propagation-defective indicator phages from Salmonella-specific TSP1 bacteriophage A Salmonella-specific propagation-defective indicator phage was constructed from a Salmonella-specific parent phage by using homologous recombination, as illustrated in Figure 13. To generate the propagation-defective indicator phage, the coding sequence for the gp22 prohead scaffold protein of the parent phage was replaced with the NANOLUC® coding sequence in wild-type TSP1. Salmonella ATCC 19585 was transformed with a homologous recombination (HR) plasmid containing the NanoLuc gene flanked by matching bacterial genomic sequences flanking gp22 (i.e., gp21 prohead core and protease, and gp23 major capsid protein) (Figure 13). The transformed bacteria were infected with the parent phage to allow homologous recombination with the HR plasmid, deleting the phage copy of gp22, thereby simultaneously creating a propagation-defective mutant, and inserting the indicator gene (i.e., NANOLUC®) to create the propagation-defective indicator phage. Infected cells produced a mix of wild-type and recombinant bacteriophage at a recombinant:wild-type phage ratio of approximately 1:8. Co-infection with wild-type phage supports recombinant replication by complementing the missing gp22 gene in trans. Following homologous recombination, a series of titering and enrichment steps were used to isolate specific recombinant bacteriophage expressing NANOLUC®. Large-scale production was performed to obtain high-titer stocks of propagation-deficient indicator phage suitable for use in detection assays. As illustrated in Figure 14, the propagation-deficient indicator phage (designated TSP1.Δgp22 NanoLuc) was unable to grow in wild-type Salmonella 19585 and was therefore propagated in an engineered Salmonella strain (a "permissive" Salmonella strain) transformed with a high-copy pUC-based plasmid expressing the gp22 prohead scaffold protein.

[0171] Isolated TSP1.Δgp22.NanoLuc plaques were suspended in TMS buffer and inoculated into either wild-type or permissive Salmonella 19585 cultures and incubated for 3 hours at 37°C. NanoGlo (PROMEGA®) assays were performed on 10 μl samples. TSP1.Δgp22.NanoLuc infection of both wild-type and permissive Salmonella resulted in a signal above background (100 RLU / sec) (Figure 15).

[0172] Example 5: Examination of the detection limit of Salmonella-specific propagation-defective TSP1 indicator phage To assess the detection limit of the TSP1.Δgp22.NanoLuc propagation-deficient indicator phage in stationary-phase Salmonella, Salmonella Typhimurium ATCC 19585 was grown for 18-20 hours until stationary phase. The stationary-phase culture was diluted in TSB, and the cells were transferred to a 96-well plate according to the plate layout shown in Figure 16. The TSP1.Δgp22.NanoLuc phage was added to the Salmonella stationary-phase culture and incubated for 2 hours at 37°C. After infection with the phage, lysis buffer, assay buffer, and substrate were added, and the plate was read in a luminometer for 1 second. The results are shown in Figure 16.

[0173] To assess the detection limit of the TSP1.Δgp22.NanoLuc propagation-deficient indicator phage in logarithmic-phase Salmonella, Salmonella Typhimurium ATCC 19585 was grown for 18–20 h until stationary phase. The stationary-phase cell culture was then diluted into TSB and grown to early logarithmic phase. The logarithmic-phase Salmonella culture was then diluted into TSB, and the cells were transferred to a 96-well plate according to the layout shown in Figure 17. The TSP1.Δgp22.NanoLuc phage was added to the logarithmic Salmonella culture and incubated for 2 h at 37°C. After infection with the phage, lysis buffer, assay buffer, and substrate were added, and the plate was read in a luminometer for 1 second. The results are shown in Figure 17.

[0174] Example 6: Creation and isolation of propagation-defective SEA1 indicator phage from Salmonella-specific bacteriophages. Using homologous recombination as illustrated in Figure 18, a Salmonella-specific propagation-defective indicator phage was constructed from a Salmonella-specific parent phage. To generate the propagation-defective indicator phage, the coding sequence for the gp84 baseplate wedge subunit protein of the parent phage was replaced with the NANOLUC® coding sequence in wild-type SEA1. Salmonella 27869 was transformed with both homologous recombination (HR) plasmids containing the NanoLuc gene flanked by matching bacterial genomic sequences flanking gp84 (i.e., the gp83 head protein and the gp85 baseplate hub subunit and tail lysozyme) (Figure 18). These transformed bacteria were then infected with the parent phage to allow homologous recombination with the HR plasmid, deleting the phage copy of gp84, thereby simultaneously creating a propagation-defective mutant, and inserting the indicator gene (i.e., NANOLUC®) to create the propagation-defective indicator phage. Infected cells produced a mix of wild-type and recombinant bacteriophage. Co-infection with wild-type phage supported recombinant replication by complementing the missing gp84 gene in trans. Following homologous recombination, a series of titering and enrichment steps were used to isolate specific recombinant bacteriophage expressing NANOLUC®. Large-scale production was performed to obtain high-titer stocks of propagation-deficient indicator phage suitable for use in detection assays. As illustrated in Figure 19, the propagation-deficient indicator phage (designated SEA1.Δgp84.NanoLuc) was unable to grow in wild-type Salmonella 27869 and was therefore propagated in an engineered Salmonella strain (a "permissive" Salmonella strain) transformed with a high-copy pUC-based plasmid expressing the gp84 baseplate wedge subunit protein.

[0175] Example 7: Examination of Reproduction-Defective SEA1 Indicator Phages Specific for Wild-Type and Permissive Salmonella Time course infection of wild-type Salmonella 27869 compared to pUC57.trans.SEA1.gp84-transformed 27869 permissive cells. 1.0 x 10 6 Either wild-type Salmonella 27869 cells / well (in 200 μl TSB) or pUC57.trans.SEA1.gp84-transformed permissive 27869 cells (in 200 μl TSB+carb) were incubated in triplicate with recombinant bacteriophage (MOI of 0.1). NanoGlo assays were performed on 10 μl samples at 37°C for 4 hours. The signal produced by the propagation-defective recombinant phage in wild-type Salmonella initially plateaued at a low level, demonstrating the lack of sustained growth of the phage in wild-type Salmonella (Figure 20). However, the signal in permissive Salmonella continued to increase over time, indicating multiple rounds of infection and continued growth (Figure 20).

[0176] Next, a time course infection of wild-type Salmonella strains 7001, 8326, 13076, and 27869 was performed. 6 Cells / well of each wild-type Salmonella strain were incubated with recombinant bacteriophage (MOI of 0.01) in 100 μl of TSB. NanoGlo assays were performed on 10 μl samples at 0, 1, 2, and 5 hours at 37°C. The signal produced by the propagation-defective recombinant phage in wild-type Salmonella initially plateaued at a low level (Figure 21).

[0177] Replication of the SEA1.Δgp84.NanoLuc bacteriophage in wild-type Salmonella strains was assessed by performing plaque assays on 5-hour wild-type cultures (40 μl cultures). No plaques formed from cultures of wild-type Salmonella strains 7001, 8326, 13076, and 27869 (FIG. 22), confirming the lack of replication of SEA1.Δgp84.NanoLuc in wild-type Salmonella strains.

[0178] Example 8: Examination of the detection limit of the Salmonella-specific propagation-defective SEA1 indicator phage To assess the detection limit of the SEA1.Δgp84.NanoLuc propagation-defective indicator phage, Salmonella Newport ATCC 27869 was transformed with AmpR puc57.SEA1.transgp84. Log-phase cultures were diluted in TSB, and cells were transferred to 96-well plates according to the plate layout shown in Figure 23. SEA1.Δgp84.NanoLuc phage was added to the Salmonella log-phase cultures and incubated for 2 hours at 37°C. After infection with the phage, lysis buffer, assay buffer, and substrate were added, and the plate was read in a luminometer for 1 second. The results are shown in Figure 23.

[0179] To assess the detection limit of the SEA1.Δgp84.NanoLuc propagation-deficient indicator phage in stationary-phase Salmonella, Salmonella chloreaesuis ATCC 27869 was grown for 18-20 hours until stationary phase. Stationary-phase cells were then diluted in TSB, and the cells were transferred to a 96-well plate according to the plate layout shown in Figure 24. SEA1.Δgp84.NanoLuc phage was added to the Salmonella stationary-phase culture and incubated for 2 hours at 37°C. After infection with the phage, lysis buffer, assay buffer, and substrate were added, and the plate was read in a luminometer for 1 second. The results are shown in Figure 24.

[0180] To assess the detection limit of the SEA1.Δgp84.NanoLuc propagation-deficient indicator phage in log-phase Salmonella, Salmonella chloreaesuis ATCC 27869 was grown for 18-20 hours until stationary phase. The stationary-phase cells were then diluted into TSB and grown to early log phase. The log-phase culture was then diluted into TSB, and the cells were transferred to a 96-well plate according to the plate layout shown in Figure 25. The SEA1.Δgp84.NanoLuc phage was added to the Salmonella stationary-phase culture and incubated for 2 hours at 37°C. After infection with the phage, lysis buffer, assay buffer, and substrate were added, and the plate was read in a luminometer for 1 second. The results are shown in Figure 25.

[0181] Example 9: Testing of Salmonella-specific, propagation-defective SEA1 indicator phage in a detection assay To evaluate the activity of the SEA1.Δgp84.NanoLuc propagation-defective indicator phage, its detection activity was compared to that of SEA1 NanoLuc, a Salmonella-specific propagation-competent indicator phage with the NANOLUC® gene inserted after gp84. Each sample was infected with either SEA1 NanoLuc or SEA1.Δgp84.NanoLuc for 2 and 4 hours at 37°C. Lysis buffer and luciferase substrate were added, and the samples were read in a luminometer. Five replicate measurements were performed at each CFU level for each phage, as shown in Figure 26A. The RLU values ​​for each CFU are shown in Figure 26B (2-hour infection) and Figure 26C (4-hour infection). The present invention provides, for example, the following items. (Item 1) A recombinant phage containing an indicator gene in the late gene region of the phage genome, which is reproduction-deficient and capable of specifically infecting a microorganism of interest. (Item 2) 2. The recombinant phage of item 1, wherein the bacteriophage is propagation-deficient due to an alteration or deletion in a late gene required for virion assembly. (Item 3) 2. The recombinant phage according to item 1, wherein the indicator gene is inserted into the sequence of the late gene of the recombinant phage, rendering the late gene non-functional and the recombinant phage propagation-defective. (Item 4) 2. The recombinant phage of claim 1, wherein the indicator gene replaces at least a portion of the sequence of a late gene of the recombinant phage, rendering the recombinant phage propagation-defective, and the late gene is required for virion assembly. (Item 5) 2. The recombinant phage according to item 1, which is derived from a phage specific for E. coli or Salmonella or Listeria or Staphylococcus. (Item 6) 2. The recombinant phage of item 1, wherein the late genes are required for virion assembly. (Item 7) A composition comprising at least two recombinant phages, each containing an indicator gene in the late gene region of the phage genome, wherein the recombinant phages are reproduction-deficient and capable of specifically infecting one or more target microorganisms. (Item 8) 8. The composition of claim 7, wherein each of the at least two recombinant phages comprises a different indicator gene. (Item 9) 9. The composition according to item 8, wherein each of the at least two recombinant phages is capable of specifically infecting a different target microorganism. (Item 10) 9. The composition of claim 8, wherein the at least two recombinant phages are capable of infecting multiple target microorganisms. (Item 11) 8. The composition according to item 7, wherein the target microorganism comprises at least one of E. coli, Salmonella, Listeria, and Staphylococcus. (Item 12) 11. The composition of claim 10, wherein the plurality of target microorganisms comprises at least two different categories of bacteria. (Item 13) 13. The composition of item 12, wherein the at least two different categories of bacteria comprise one or more of at least two different genera of bacteria, at least two different species of bacteria, at least two different strains of bacteria, or at least two different serotypes of bacteria. (Item 14) 1. A method for preparing a recombinant phage, comprising: selecting parent phages that specifically infect the target microorganism; genetically modifying the parent phage to generate a recombinant, propagation-defective phage; transforming the engineered strain of the target microorganism capable of expressing the product of the gene mutated in the propagation-defective phage with a homologous recombination (HR) plasmid containing an indicator gene and HR sequences flanking the indicator gene that are homologous to a desired sequence in the parent phage; infecting the transformed target microorganism with the parent phage or the propagation-defective parent phage to allow HR to occur between the HR plasmid and the genome of the parent phage or the recombinant propagation-defective phage; isolating specific clones of recombinant phage that are reproduction-deficient and capable of expressing the product of said indicator gene; A method comprising: (Item 15) Item 15. The method according to item 14, wherein the step of altering the gene of the parent phage to generate the propagation-defective phage is achieved by the HR occurring between the HR plasmid and the genome of the parent phage, and the gene of the parent phage is altered by replacement of at least a portion of the parent phage with the indicator gene. (Item 16) 15. The method of claim 14, further comprising generating the engineered strain of the target microorganism. (Item 17) 17. The method of claim 16, wherein generating the engineered strain of the target microorganism comprises transforming the target microorganism with a plasmid encoding and capable of expressing the gene that has been altered in the recombinant, propagation-defective phage. (Item 18) 15. The method of claim 14, wherein transforming the engineered strain further comprises transforming the engineered strain with a trans-plasmid. (Item 19) 15. The method according to item 14, further comprising the step of preparing the homologous recombination plasmid containing the indicator gene prior to the transforming step. (Item 20) 15. The method of claim 14, wherein the step of isolating the specific clones of recombinant phage that are reproduction-deficient and capable of expressing the product of the indicator gene comprises performing a limiting dilution assay to isolate clones that exhibit expression of the indicator gene. (Item 21) 15. The method of claim 14, wherein the recombinant phage is derived from a phage specific for E. coli, Salmonella, Listeria, or Staphylococcus. (Item 22) 1. A method for detecting a microorganism of interest in a sample, comprising: Incubating the sample with the recombinant phage according to item 1; detecting a product of an indicator gene, wherein positive detection of said product of said indicator gene indicates that said microorganism of interest is present in said sample; A method comprising: (Item 23) 23. The method of claim 22, wherein the sample is a food sample, an environmental sample, a water sample or a commercial sample. (Item 24) 23. The method of claim 22, wherein as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single microorganism is detected in the sample. (Item 25) 23. The method according to item 22, wherein the target microorganism is E. coli, Salmonella, Listeria, or Staphylococcus. (Item 26) 23. The method according to item 22, wherein the target microorganism is Salmonella. (Item 27) A kit for detecting a microorganism of interest in a sample, comprising the recombinant phage according to item 1 and a substrate for reacting with a product of an indicator gene to detect the product of the indicator gene. (Item 28) A system for detecting a microorganism of interest, comprising the recombinant phage according to item 1 and components for detecting the product of the indicator gene.

Claims

[Claim 1] The invention described in the specification.

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