Devices and methods for detecting microorganisms using recombinant reproductive-deficient indicator bacteriophages

Abstract

Disclosed herein are compositions, methods, kits, and systems for the rapid detection of microorganisms using a reproductive-deficient indicator bacteriophage. The specificity of such reproduction-deficient indicator bacteriophages to bind and infect specific microorganisms of interest allows targeted and sensitive detection of microorganisms of interest.

Description

[0001] This application is a divisional application of the same invention patent application 202080060223.0, filed on August 26, 2020.

[0002] [Incorporated via reference]

[0003] This application claims priority to U.S. Provisional Application No. 62 / 891,701, filed August 26, 2019. The disclosures of the following U.S. patent applications are incorporated herein by reference in their entirety: U.S. Application No. 16 / 247,490, filed January 14, 2019; U.S. Patent Application No. 16 / 247,486, filed January 14, 2019; U.S. Application No. 16 / 298,695, filed March 11, 2019; U.S. Provisional Application No. 62 / 640,793, filed March 9, 2018; U.S. Provisional Application No. 62 / 798,980, filed January 30, 2019; U.S. Application No. 13 / 773,339, filed February 21, 2013; and U.S. Application No. 1, filed February 18, 2015. U.S. Application No. 4 / 625,481, filed September 13, 2016; U.S. Application No. 15 / 263,619, filed January 18, 2017; U.S. Application No. 15 / 409,258, filed January 12, 2018; U.S. Provisional Application No. 62 / 616,956, filed January 12, 2018; U.S. Provisional Application No. 62 / 628,616, filed February 9, 2018; U.S. Provisional Application No. 62 / 661,739, filed April 24, 2018; U.S. Provisional Application No. 62 / 640,793, filed March 9, 2018; and U.S. Provisional Application No. 62 / 798,980, filed January 30, 2019. [Field of Invention]

[0004] This disclosure relates to methods, apparatus, and systems for detecting microorganisms of interest using recombinant infectious agents. [Background Technology]

[0005] There is a strong interest in improving the speed and sensitivity of detecting bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause extremely high rates of morbidity in humans and livestock, as well as enormous economic losses. Given the outbreaks of life-threatening or fatal illnesses caused by the ingestion of food contaminated with certain microorganisms (e.g., 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 and Prevention (CDC).

[0006] Traditional microbiological assays for detecting bacteria rely on non-selective and selective enrichment cultures, followed by inoculation onto selective media and further testing to confirm suspected colonies. Such procedures can take several days. A variety of rapid methods have been investigated and put into practice to reduce time requirements. However, to date, these time-reducing methods have some drawbacks. For example, techniques involving direct immunoassays or gene probes generally require an overnight enrichment step to achieve sufficient sensitivity, thus lacking the ability to provide results on the same day. Polymerase chain reaction (PCR) assays also include an amplification step, thus enabling very high sensitivity and selectivity; however, the sample size that can be economically accepted for PCR testing is limited. Diluted bacterial suspensions suitable for PCR must be cell-free, thus still requiring purification and / or lengthy enrichment steps.

[0007] The time required for traditional bioaccumulation is determined by the growth rate of the target bacterial population in the sample, the role of the sample matrix, and the required sensitivity. In practice, most high-sensitivity methods employ overnight incubation, taking approximately 24 hours in total. Due to the time required for cultivation, these methods can take up to three days, depending on the organism being identified and the source of the sample. This lag time is generally undesirable because such delays allow contaminated food, water, or other products to enter livestock or humans. Furthermore, the increase in antibiotic-resistant bacteria and biodefense considerations make rapid identification of bacterial pathogens in water, food, and clinical samples a critical priority worldwide.

[0008] Therefore, there is a need for faster, simpler, and more sensitive methods for detecting and identifying microorganisms, such as bacteria and other potentially pathogenic microorganisms. [Summary of the Invention]

[0009] Embodiments of this disclosure include devices, compositions, methods, apparatus, systems, and kits for detecting microorganisms (such as, but not limited to, bacteria). This disclosure can be implemented in a wide variety of ways. Some exemplary embodiments of this application are discussed below.

[0010] An exemplary embodiment of this disclosure is a recombinant phage containing an indicator gene in a late gene region of the phage's genome, wherein the recombinant phage is reproductively defective and wherein the recombinant phage is capable of specifically infecting microorganisms of interest. In some embodiments, the recombinant phage becomes reproductively defective due to alterations to the late gene required for viral particle assembly. In some embodiments of the recombinant phage, the indicator gene is inserted into the sequence of the late gene of the recombinant phage, rendering the late gene nonfunctional and the recombinant phage reproductively defective. In some embodiments of the recombinant phage, the indicator gene replaces at least a portion of the sequence of the late gene of the recombinant phage, rendering the recombinant phage reproductively defective, wherein the late gene is required for viral particle assembly. The recombinant phage is derived from a phage specific to *Escherichia coli*, *Salmonella*, *Listeria*, or *Staphylococcus*. In some embodiments, the recombinant phage is derived from a phage specific to *Escherichia coli*. In other embodiments, the recombinant phage is derived from a phage specific to the genus Salmonella. In some embodiments of the recombinant phage, the late gene is required for viral particle assembly.

[0011] Exemplary embodiments of this disclosure are compositions comprising at least two recombinant phages, each of which contains an indicator gene in a late gene region of its genome, wherein the recombinant phage is reproductively defective and capable of specifically infecting one or more microorganisms of interest. In some embodiments of the composition, each of the at least two recombinant phages contains a different indicator gene. In some embodiments of the composition, each of the at least two recombinant phages is capable of specifically infecting different microorganisms of interest. In some embodiments of the composition, the at least two recombinant phages are capable of infecting multiple microorganisms of interest. In some embodiments of the composition, the multiple microorganisms of interest comprise at least two different classes of bacteria. In some embodiments of the composition, the at least two different classes of bacteria comprise one or more of at least two different genera, at least two different species, at least two different strains, or at least two different serotypes of bacteria.

[0012] An exemplary embodiment of this disclosure is a method for preparing recombinant phages. This method may include the following steps: selecting a parental phage specifically to infect a target microorganism; altering the genes of the parental phage to produce a recombinant reproductively deficient phage; transforming an engineered strain of the target microorganism with a homologous recombination (HR) plasmid, the engineered strain being capable of expressing the product of a mutated gene in the reproductively deficient phage, the homologous recombination (HR) plasmid comprising an indicator gene and an HR sequence flanking the indicator gene and homologous to a desired sequence in the parental phage; infecting the transformed target microorganism with the parental phage or the reproductively deficient parental phage such that HR occurs between the HR plasmid and the genome of the parental phage or the recombinant reproductively deficient phage; and isolating specific clones of the recombinant phage, the specific clones being reproductively deficient and capable of expressing the product of the indicator gene. In some embodiments of the method for preparing recombinant phages, the alteration of the parental phage's genes to produce the reproductively defective phage is achieved through an HR (human vegetative-transfer) process occurring between the HR plasmid and the genome of the parental phage, wherein the parental phage's genes are altered by replacing at least a portion of the parental phage with the indicator gene. In some embodiments, altering the genes includes partially or completely deleting the genes of the parental phage. Thus, in some embodiments, the method includes altering the genome of the parental phage, wherein at least one gene of the parental phage is deleted. In some embodiments, at least two, three, four, or five genes are deleted.

[0013] Some embodiments of the method for preparing recombinant phages may further include the step of generating an engineered strain of the target microorganism. In some embodiments, the step of generating an engineered strain of the target microorganism may include transforming the target microorganism using a plasmid (“trans plasmid”) that encodes a gene altered in the recombinant reproductively deficient phage. Some embodiments of the method for preparing recombinant phages may further include the step of preparing a homologous recombinant plasmid containing the indicator gene prior to the transformation step. In some embodiments, the step of generating an engineered strain of the target microorganism may include transforming the target microorganism using a trans plasmid and an HR plasmid containing the indicator gene. In some embodiments of the method for preparing recombinant phages, altering the genes of a parental phage to generate a reproductively defective phage is achieved by infecting a wild-type parental phage of an engineered target microorganism containing a trans plasmid and an HR plasmid, such that an HR can occur between the HR plasmid and the genome of the parental phage, wherein the genes of the parental phage are altered by replacing at least a portion of the parental phage with the indicator gene, and a plasmid (trans plasmid) containing the gene altered in the reproductively defective recombinant phage provides the trans gene, complementing the gene deletion or alteration in the reproductively defective phage. In a further embodiment of the method for preparing recombinant phages, gene deletion of a parental phage to generate a reproductively defective phage is achieved by infecting a wild-type parental phage of an engineered target microorganism containing a trans plasmid and an HR plasmid, such that HR can occur between the HR plasmid and the genome of the parental phage, wherein the genome of the parental phage is altered by replacing at least a portion of the parental phage with the indicator gene, and a plasmid (trans plasmid) containing the gene altered in the reproductively defective recombinant phage provides the trans gene, complementing the gene deletion or alteration in the reproductively defective phage.

[0014] In some embodiments of the method for preparing recombinant phages, altering the genes of a parental phage to generate a reproductively defective phage is achieved by infecting a wild-type parental phage of an engineered target microorganism containing an HR plasmid instead of a plasmid that encodes and is capable of expressing the altered gene in the reproductively defective phage. This allows HR to occur between the HR plasmid and the genome of the parental phage, wherein the genes of the parental phage are altered by replacing at least a portion of the parental phage with the indicator gene, and the wild-type parental phage infecting or co-infecting bacteria trans-provides the gene, complementing the gene deletion or alteration in the reproductively defective phage.

[0015] In some embodiments, the step of isolating specific clones of recombinant phages may include performing limiting dilution assays to isolate clones demonstrating expression of an indicator gene, said specific clones being reproductively defective and capable of expressing said indicator gene. The recombinant phages are derived from phages specific to *Escherichia coli*, *Salmonella*, *Listeria*, or *Staphylococcus*. In some embodiments of the method for preparing recombinant phages, the recombinant phages are derived from phages specific to *Escherichia coli*. In some embodiments of the method for preparing recombinant phages, the recombinant phages are derived from phages specific to *Salmonella*.

[0016] An exemplary embodiment of this disclosure is a method for detecting a microorganism of interest in a sample, comprising the steps of: incubating the sample with a recombinant bacteriophage as described in an embodiment of this disclosure; and detecting the product of the indicator gene, wherein a positive detection of the product of the indicator gene 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*.

[0017] Exemplary embodiments of this disclosure also include a kit for detecting a microorganism of interest in a sample, the kit comprising a recombinant phage according to an embodiment of this disclosure and a substrate for reacting with the product of an indicator gene to detect the product of the indicator gene. Exemplary embodiments of this disclosure also include a system for detecting a microorganism of interest, the system comprising the recombinant phage of claim 1 and a component for detecting the product of the indicator gene. [Attached Image Description]

[0018] This disclosure can be better understood by referring to the following non-limiting drawings.

[0019] Figure 1 An exemplary method for preparing recombinant reproductive defect indicator phages is schematically illustrated, wherein the introduction of reproductive defect and indicator genes into parental phages is completed in a one-step recombination process.

[0020] Figure 2 The illustration schematically depicts plasmid transformation using genes expressing those required for phage replication and uses indicators of reproductive defects to demonstrate "permissive" microorganisms susceptible to phage infection.

[0021] Figure 3 The diagram schematically illustrates the generation of a reproductively defective indicator phage CBA120.Δgp22.NanoLuc through homologous recombination via trans-complementation using co-infection with a CBA120 Escherichia coli-specific phage.

[0022] Figure 4 This table shows the detection limits of indicator phages CBA120.Δgp22.NanoLuc that are reproductively defective in stationary Escherichia coli O157:H7 ATCC 43888.

[0023] Figure 5 The illustration schematically depicts the propagation of a propagation-deficient indicator phage specific to the E. coli O157:H7 serotype in an engineered E. coli O157:H7 strain (“permissive” E. coli O157:H7 strain) transformed with a plasmid expressing gp22 prohead scaffold protein.

[0024] Figure 6 Exemplary growth curves of indicator phages with reproductive defects are shown, in which the phages were successfully grown in permissible Escherichia coli strain O157:H7.

[0025] Figure 7 The strategy of using a reproductively defective indicator phage specific to Escherichia coli O157:H7 serotype is illustrated schematically.

[0026] Figure 8 This is a bar graph illustrating the raw signal results of detection using a reproductively defective indicator phage compared to a replicable indicator phage specific to the E. coli O157:H7 serotype during the logarithmic phase.

[0027] Figure 9 This is a bar graph illustrating the signal-to-background ratio of the detection results using a reproductively defective indicator phage compared to the results obtained using a replicable indicator phage specific to the E. coli O157:H7 serotype during the logarithmic growth phase.

[0028] Figure 10 This is a bar graph illustrating the raw signal results of detection using a reproductively defective indicator phage compared to a replicable indicator phage specific to the E. coli O157:H7 serotype during the stationary phase of E. coli O157:H7.

[0029] Figure 11This is a bar graph illustrating the signal-to-background ratio of the detection results using a reproductively defective indicator phage compared to the results obtained using a replicable indicator phage specific to the E. coli O157:H7 serotype during the stationary phase of E. coli O157:H7.

[0030] Figure 12 This is a line graph illustrating the specificity of indicator phages that exhibit reproductive defects specific to Escherichia coli O157:H7 serotype.

[0031] Figure 13 The diagram schematically illustrates the generation of a reproductively defective indicator phage TSP1.Δgp22.NanoLuc through homologous recombination via trans-complementation of co-infection with a TSP1 Salmonella-specific phage.

[0032] Figure 14 The illustration schematically depicts the propagation of a Salmonella-specific recombinant reproductive defective indicator phage in an engineered Salmonella strain (“permissive” Salmonella strain) transformed with a plasmid expressing the gp22 head scaffold protein.

[0033] Figure 15 This is a bar graph illustrating the raw signal results of detection assays using reproductively defective indicator phages in wild-type Salmonella compared to permissible Salmonella.

[0034] Figure 16 This table shows the detection limits of indicator phages that are reproductively defective in Salmonella typhimurium (ATCC 19585) SP1.Δgp22.NanoLuc during the stationary phase.

[0035] Figure 17 This table shows the detection limits of indicator phages that are reproductively defective in Salmonella Typhimurium ATCC 19585 during the logarithmic growth phase, specifically SP1.Δgp22.NanoLuc.

[0036] Figure 18 The diagram schematically illustrates the generation of a reproductively defective indicator phage SEA1.Δgp84.NanoLuc through homologous recombination via trans-complementation co-infection with a Salmonella-specific phage.

[0037] Figure 19 The illustration schematically depicts the propagation of a Salmonella-specific recombinant reproductive defective indicator phage in an engineered Salmonella strain (“permissive” Salmonella strain) transformed with a plasmid expressing the gp84 substrate wedge protein.

[0038] Figure 20This is a line graph illustrating the raw signal results of the detection of the indicator phage SEA1.Δgp84.NanoLuc in wild-type Salmonella compared to permissive Salmonella.

[0039] Figure 21 This is a line graph illustrating the raw signal results of detection using SEA1.Δgp84.NanoLuc, an indicator phage for reproductive defects in wild-type Salmonella strains 7001, 8326, 13076, and 27869.

[0040] Figure 22 This is a bar graph illustrating plaque assays of the replication of SEA1.Δgp84.NanoLuc, an indicator phage, in wild-type Salmonella strains 7001, 8326, 13076, and 27869, demonstrating the reproductive defects of SEA1.Δgp84.NanoLuc.

[0041] Figure 23 This table shows the detection limits of indicator phages that demonstrate the reproductive defects of SEA1.Δgp84.NanoLuc in Salmonella newport ATCC 27869 transformed with AmpR pUC57 SEA1.Trans gp84 during the logarithmic growth phase.

[0042] Figure 24 This table shows the detection limits of indicator phages that are reproductively defective in Salmonella chloreaesuis ATCC 7001 during the stationary phase.

[0043] Figure 25 This table shows the detection limits of indicator phages that are reproductively defective in *Salmonella choleraesuis* ATCC 7001 during the logarithmic growth phase.

[0044] Figure 26A This is a table illustrating the approximate number of phages replicated by SEA1.NanoLuc and the number of CFUs that did not replicate by SEA1.Δgp84.NanoLuc per well. Figure 26B This table shows the RLU signal results at 2 hours post-infection, comparing the replicated phage SEA1.NanoLuc, which is specific to Salmonella Typhimurium, with those of the replicated phage and the indicator phage SEA1.Δgp84.NanoLuc, which is a replication defective phage. Figure 26C This table shows the RLU signal results at 4 hours post-infection, comparing the replicated phage SEA1.NanoLuc, which is specific to Salmonella Typhimurium, with those of the replicated phage and the indicator phage SEA1.Δgp84.NanoLuc, which is a replication defective phage.

[0045] Figure 27 The method described is to identify reproductively defective indicator phages by isolating recombinant reproductively defective indicator phages using a series of sequential infection and dilution steps.

[0046] Figure 28 The present disclosure describes an embodiment of detecting microorganisms of interest by detecting luciferase using a recombinant reproductively deficient indicator phage encoding a soluble luciferase.

[0047] Figure 29 A filter plate assay according to an embodiment of the present disclosure is described for detecting microorganisms of interest using recombinant reproductively deficient indicator phages, wherein the microorganism of interest and the recombinant reproductively deficient indicator phages are incubated on a filter plate and the indicator protein is detected directly without removing the incubation medium.

[0048] Figure 30 An embodiment of the present disclosure is described as a “concentration-free assay” for detecting microorganisms of interest using recombinant reproductively defective indicator phages.

[0049] Figure 31 The present disclosure describes a hybrid immunophage (HIP) assay for detecting microorganisms of interest using a recombinant reproductively deficient indicator phage, wherein an antibody against the microorganism of interest is used to capture the microorganism on the surface of the assay well prior to incubation with the recombinant reproductively deficient indicator phage.

Detailed Implementation Methods

[0050]

definition

[0051] Unless otherwise specified herein, the scientific and technical terms used in connection with this invention shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature used herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as the techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, are those well-known and commonly used in the art. Unless otherwise specified, known methods and techniques are generally performed according to conventional methods well-known in the art and as described in various general literatures and more specific literatures discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature used in connection with the laboratory operations and techniques described herein is those well-known and commonly used in the art.

[0052] Unless otherwise specified, the following terms shall be understood to have the following meanings:

[0053] As used herein, the terms “a,” “an,” and “the” may refer to one or more, unless specifically otherwise indicated.

[0054] The term “or” is used to mean “and / or” unless explicitly stated otherwise, indicating that it refers to only one or that the alternatives are mutually exclusive, although this disclosure supports the definition of only one and “and / or”. As used herein, “another” may mean at least a second or more.

[0055] Throughout this application, the term "about" is used to refer to a numerical value that includes variations in the inherent error of the apparatus or method used to determine the value, or variations present in the sample.

[0056] The term "solid support" or "support" refers to a structure on which a substrate and / or surface can bind biomolecules. For example, a solid support can be a measurement well (i.e., such as a microtiter plate or multi-well plate), or a solid support can be a location on a filter, array, or movable support such as beads or membranes (e.g., filter plates or side-flow test strips).

[0057] The term "binding agent" refers to a molecule that can specifically and selectively bind a second (i.e., a different) molecule of interest. The interaction can be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it can be covalent. The term "soluble binding agent" refers to a binding agent that does not associate with a solid support (i.e., covalent or non-covalent binding).

[0058] As used herein, the terms "reproductive defective" or "replication defective" refer to an impairment of a bacteriophage's reproductive capacity. That is, a reproductively defective bacteriophage may be unable to generate new bacteriophage particles, for example, due to a lack of proteins required for capsid assembly. Various deletions, insertions, or substitutions in the bacteriophage genome can render a bacteriophage reproductively defective.

[0059] As used herein, "analyte" refers to the molecule, compound, or cell being measured. In some embodiments, the analyte of interest may interact with a binding agent. As described herein, the term "analyte" may refer to the protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may not have a biological effect. Analytes may include small molecules, carbohydrates, oligosaccharides, lipids, peptides, peptide mimics, organic compounds, etc.

[0060] The terms "detectable moiety," "detectable biomolecule," "reporter," "indicator," or "indicator portion" refer to a molecule or compound produced by a molecule (such as an enzyme) that can be measured in a quantitative assay. For example, an indicator or indicator portion may include an enzyme that can be used to convert a substrate into a measurable product. An indicator or indicator portion may be an enzyme that catalyzes a reaction that produces bioluminescent emission (e.g., luciferase). Alternatively, an indicator or indicator portion may be a quantifiable radioisotope. Alternatively, an indicator or indicator portion may be a fluorophore. Alternatively, other detectable molecules may be used. The term "indicator gene" is used to refer to a gene that encodes an indicator, such as a protein, for example, an enzyme.

[0061] As used herein, "bacteriophage" includes one or more viruses capable of invading live bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms. In this disclosure, the term "bacteriophage" and related terms include viruses such as: bacterial bacteriophages capable of invading bacteria and archaea; mycobacterial bacteriophages capable of invading mycobacteria (bacterial families, including mycobacteria of the Mycobacterium tuberculosis complex, including pathogens of tuberculosis, and mycobacteria of the Mycobacterium avis complex, including pathogens of tuberculosis); fungal bacteriophages capable of invading fungi; mycoplasma bacteriophages; and viruses capable of infecting protozoa, yeast, and other microscopic living organisms. Here, "microscopic" means a maximum size of 1 millimeter or less. Bacteriophages are viruses that have evolved in nature to use bacteria, mycobacteria, or archaea as tools for replicating themselves. In nature, bacteriophages attach themselves to microorganisms and inject their DNA (or RNA) into them, which can then induce the microorganisms to replicate the phage hundreds or even thousands of times. This is called phage amplification. For example, well-studied *E. coli* phages include T1, T2, T3, T4, T5, T7, and λ; other *E. coli* phages available at the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. *Salmonella* phages include TSP1, TSP11, SPN1S, 10, ε15, SEA1, TSP1, and P22. *Listeria* phages include P100, LMA8, LMA4, LPES1, LipZ5, P40, vB_LmoM_AG20, P70, P100, LP-JS3, LP-ES1, and A511. Staphylococcal phages include Staphylococcus ISP, P4W, Virus K, Twort, phi11, 187, P68, and phiWMY.

[0062] As used herein, the “late gene region” refers to a region of the viral genome transcribed late in the viral life cycle. The late gene region typically includes the most abundantly expressed genes (e.g., structural proteins that assemble into phage particles). Late genes in phages are synonymous with class III genes and include genes with both structural and assembly functions. For example, late genes (synonymous with class III genes) are transcribed in phage T7, for instance, from 8 minutes post-infection until lysis, class I genes (e.g., RNA polymerase) earlier from 4 to 8 minutes, and class II genes from 6 to 15 minutes; thus, there is an overlap in the timing of classes II and III. Late promoters are naturally located and active within this late gene region.

[0063] As used herein, "culture for enrichment" refers to conventional culture, such as incubation in a medium that promotes the growth of microorganisms, and should not be confused with other possible uses of the term "enrichment," such as enrichment by removing the liquid components of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not include conventional methods of promoting microbial growth. In some embodiments of the methods described herein, a culture for enrichment may be employed for a sustained period of time.

[0064] As used herein, “recombination” refers to gene (i.e., nucleic acid) modification, such as that typically performed in the laboratory to combine genetic material that would not otherwise be found together. This term is used interchangeably with the term “modified” as used herein. As used herein, “RLU” refers to relative optical units, such as those measured by a photometer (e.g., [photometer name missing]). 96) or similar instruments for measuring light. For example, the reaction between luciferase and a suitable substrate (e.g., The detection of NanoGlo is often reported as the RLU detected.

[0065] [Overview]

[0066] This article discloses compositions, methods, and systems that demonstrate surprising sensitivity in the detection of microorganisms of interest (such as bacteria and archaea) in test samples (e.g., biological samples, food samples, water samples, and environmental samples). Some non-limiting examples of microorganisms of interest include: *Bacillus* spp., *Bordetella pertussis*, *Brucella* spp., *Camplylobacters* spp. (such as *Campylobacter jejuni*), *Chlamydia pneumoniae*, *Cronobacter* spp., *Clostridium perfringens*, *Clostridium botulinum*, *Enterobacter* spp., *Escherichia* spp. (such as *Escherichia coli*, e.g., *Escherichia coli* O157:H7 and other Shiga toxin-producing and enterotoxin-producing strains of *Escherichia coli*), and *Klebsiella pneumoniae*. Klebsiella pneumoniae, Klebsiella oxytoca, Listeria (such as Listeria monocytogenes), Mycoplasma pneumoniae, Pseudomonas, Salmonella (e.g., Salmonella typhi, Salmonella typhimurium, or Salmonella enteritidis), Shigella sonnei, Yersinia, Vibrio, Staphylococcus (e.g., Staphylococcus aureus), and Streptococcus. The assay can be performed within a shorter timeframe than previously thought possible using genetically modified phages in assays without utilizing cultures for enrichment, or in some implementations with minimal incubation time (during which the microorganisms can potentially multiply). It is also surprising that potentially high multiples of infection (MOI), or high concentrations of plaque-forming units (PFU), were successfully used for incubation with the test sample. Such high phage concentrations (PFU / mL) were previously considered unfavorable for bacterial detection assays because they were thought to cause “external lysis.”However, high concentrations of phages can facilitate the discovery, binding, and infection of low numbers of target cells.

[0067] The compositions, methods, systems, and kits of the present invention may comprise recombinant bacteriophages for detecting microorganisms of interest. In some embodiments, the present invention may include compositions comprising recombinant bacteriophages having an indicator gene inserted into a late gene region of the bacteriophage. Such recombinant bacteriophages are referred to as "indicator phages." In some embodiments, expression of the indicator gene after infection of a host microorganism results in the production of a soluble indicator protein product. In some embodiments, the indicator gene may be inserted into a late gene (i.e., class III) region of the bacteriophage. The recombinant phage according to embodiments of the present invention can be derived from podoviruses such as T7 and T7-like, myoviruses such as T4 and T4-like, siphoviruses such as T5, P70, and Saka6, and related phages, ViI and ViI-like (or Vi1 virus, according to GenBank / NCBI), Kronobacter spp.-specific phages such as Saka2 or Saka4, Salmonella spp. phage SPN1S, Salmonella spp. phage 10, and Salmonella spp. phages. Bacterial cells ε15, Salmonella spp. phage SEA1, Salmonella spp. phage Spn1s, Salmonella spp. phage P22, Listeria spp. phage LipZ5, Listeria spp. phage P40, Listeria spp. phage vB_LmoM_AG20, Listeria spp. phage P70, Listeria spp. phage A511, Staphylococcus spp. phage P4W, Staphylococcus spp. phage K, Staphylococcus spp. phage Twort, Staphylococcus spp. phage SA97, Escherichia coli O157:H7 phage CBA120, or another wild-type or engineered phage.

[0068] Indicator phages according to embodiments of the present invention are reproductively deficient, meaning they cannot reproduce effectively or at all after infecting the detected microorganism of interest. Reproductive deficient indicator phages according to embodiments of the present invention become reproductively deficient due to alterations to one or more suitable genes (e.g., late genes required for viral particle assembly). In some embodiments, reproductive deficient indicator phages according to embodiments of the present invention become reproductively deficient independently of the introduction of an indicator gene by introducing a mutation into a suitable gene. In some other embodiments, reproductive deficient indicator phages according to embodiments of the present invention become reproductively deficient by replacing at least a portion of a suitable gene with an indicator gene. Reproductive deficient indicator phages according to embodiments of the present invention can reproduce or replicate in a host microorganism engineered to produce the product of a mutated gene required for phage reproduction. Such engineered microorganisms are referred to as "permissive."

[0069] Reproduction-deficient indicator phages offer several advantages over previously documented indicator phages. Because reproduction-deficient indicator phages require specially engineered microorganisms for propagation, they limit the possibility of inexperienced and / or untrained suppliers producing and distributing them. The production and distribution of contaminated, low-quality reagents is a serious problem in diagnostics. By restricting the production and distribution of indicator phages to entities with certain qualifications and meeting specific standards (e.g., through official certification processes), the risk of producing low-quality or contaminated indicator phages and distributing them to diagnostic operators is reduced. Furthermore, since they cannot reproduce in host microorganisms present in the environment, reproduction-deficient indicator phages eliminate the risk of standardized diagnostic reagents containing defined concentrations and / or amounts of indicator phages being contaminated by host microorganisms before diagnostic procedures are performed. Contamination can lead to an undetectable increase in the concentration or amount of indicator phage in the reagent, and thus inaccurate test data. This problem is particularly important in quantitative or semi-quantitative assays, where the concentration or amount of indicator phage used is correlated with the intensity of the detected signal. Furthermore, because they cannot reproduce in the microorganism of interest during the diagnostic process, the reproduction-deficient indicator phages according to embodiments of the invention allow for more accurate quantitative or semi-quantitative detection of the microorganism of interest in a sample. The improved accuracy stems from the ability to control the amount of reproduction-deficient indicator phages present in the sample throughout the detection process. Since no new viable indicator phages are generated during the detection process, only the initially used reproduction-deficient indicator phage is able to express the indicator gene product post-infection. The deletion and substitution of late genes compared to early genes ensure high expression of the indicator gene, due to the inherently high expression level of late genes and the fact that deletion of early genes often results in the genome not replicating, reducing the copy number of the indicator gene per cell. Post-infection reproduction of the indicator phage can introduce significant variability into the amount of indicator signal generated during the diagnostic process. Therefore, using reproduction-deficient indicator phages according to embodiments of the invention makes it easier to standardize quantitative and semi-quantitative detection, improving the accuracy of detection results.

[0070] In some aspects, the present invention includes a method for detecting a microorganism of interest. The method may use a bacteriophage to detect the microorganism of interest. Therefore, in some embodiments, the method may include detecting the microorganism of interest in a sample by incubating the sample with a recombinant reproductively deficient indicator phage infecting the microorganism of interest. In some embodiments, the recombinant reproductively deficient indicator phage is a bacteriophage. In some embodiments, an indicator gene may be inserted into a late gene region of the phage such that expression of the indicator gene after infection of a host microorganism results in the production of an indicator gene product. The method may include detecting the indicator gene product, wherein a 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.

[0071] In some embodiments, the invention may include a system. The system may comprise at least some of the compositions of the invention. The system may also include at least some of the components for performing the method. In some embodiments, the system is configured as a kit. Thus, in some embodiments, the invention may include a system for rapidly detecting a microorganism of interest in a sample, comprising: components for incubating the sample with a reproductively deficient indicator phage specific to the microorganism of interest, wherein the reproductively deficient indicator phage contains an indicator gene; and components for detecting the indicator. In other embodiments, the invention includes software for use with the method or system.

[0072] Some embodiments of the present invention address a need in the field of microbial detection by using a phage-based method to amplify detectable signals indicating the presence of bacteria. In some embodiments, as few as a single bacterium can be detected. The principles applied herein can be applied to the detection of a wide variety of microorganisms. Because of the numerous binding sites on the surface of microorganisms available for phages and the potential for high expression levels of encoded indicators, these indicators can be more easily detected than the microorganisms themselves. In this way, embodiments of the present invention can achieve significant signal amplification from even a single infected cell.

[0073] Some embodiments of the invention disclosed and described herein utilize the fact that a single microorganism can bind to multiple recombinant reproductively deficient indicator phages according to embodiments of the invention. Upon infection by the recombinant reproductively deficient indicator phages, they are detected by an indicator encoded by the recombinant reproductively deficient indicator phage and expressed in the microorganism. This principle allows for the amplification of indicator signals from one or more cells based on the specific recognition of microbial surface receptors. For example, by exposing even a single bacterial cell to multiple reproductively deficient indicator phages, and subsequently expressing the encoded indicator gene product, the indicator signal is amplified, allowing the microorganism of interest to be detected with high sensitivity. For example, a single bacterium present in a sample can be detected using embodiments of the invention. Embodiments of the invention utilize the high specificity of phages binding to specific microorganisms as a means of detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the invention utilizes the high specificity of reproductively deficient indicator phages.

[0074] Implementations of the methods and systems of this invention can be applied to the detection and quantification of a wide variety of microorganisms (such as, but not limited to, bacteria and archaea) in a wide variety of environments, including but not limited to the detection of pathogens from food, water, and commodity samples. The methods of this invention provide high detection sensitivity and specificity, as well as rapid detection.

[0075] sample

[0076] Each embodiment of the compositions, methods, kits, and systems of the present invention allows for the rapid detection and / or quantification of microorganisms of interest in samples. For example, methods according to embodiments of the present invention can be performed in a shortened time period and yield better results.

[0077] Microorganisms detectable in samples using embodiments of the present invention include, but are not limited to, bacteria that are pathogens transmitted through food or water. Bacteria detectable by the present invention include, but are not limited to: *Bacillus* spp., *Bordedia pertussis*, *Brucella* spp., *Campylobacter* spp. (such as *Campylobacter jejuni*), *Chlamydia pneumoniae*, *Cronobacter* spp., *Clostridium perfringens*, *Botulinum toxin*, *Enterobacter* spp., *Escherichia* spp. (such as *Escherichia coli*, e.g., *Escherichia coli* O157:H7 and other Shiga toxin-producing and enterotoxin-producing strains of *Escherichia coli*), *Klebsiella pneumoniae*, *Listeria* spp. (such as *Listeria monocytogenes*), *Mycoplasma pneumoniae*, *Salmonella* spp. (e.g., *Salmonella typhi*, *Salmonella typhimurium*, or *Salmonella enteritidis*), *Shigella sonnei*, *Yersinia* spp., *Vibrio* spp., *Staphylococcus* spp. (e.g., *Staphylococcus aureus*), and *Streptococcus* spp.

[0078] Samples can be, but are not limited to, environmental samples, food samples, or water samples. Some embodiments may include medical or veterinary samples. Samples can be liquid, solid, or semi-solid. Samples can be swabs from solid surfaces. Samples can include environmental materials, such as water samples, or filters from air samples, or aerosol samples from cyclone collectors. Samples can be samples of fish, meat (such as beef, pork, or lamb, poultry), processed foods, peanut butter, infant formula, milk powder, tea, starch, eggs, milk, cheese, or other dairy products. Medical or veterinary samples include, but are not limited to, blood, saliva, cerebrospinal fluid, fecal samples, and irrigation solutions. In some embodiments, irrigation is used to collect biological samples. Irrigation is the flow of a solution (e.g., saline) through an open wound or implanted prosthesis. Therefore, in some embodiments, biological samples are wound irrigation solutions or prosthesis irrigation solutions. In some embodiments, samples can be different types of swabs.

[0079] In some embodiments, the sample can be used directly in the detection method according to embodiments of the invention without preparation, concentration, or dilution. For example, liquid samples, including, but not limited to, milk and juice, can be directly measured. In other embodiments, the sample can be diluted or suspended in a solution, which may include, but is not limited to, buffer solutions or bacterial culture media. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing, or immersing the solid in a liquid. In some embodiments, the sample should be maintained within a pH range that promotes the attachment of recombinant phages to host bacterial cells. In some embodiments, a preferred pH range may be one suitable for phage attachment to bacterial cells. The sample should also contain suitable concentrations of divalent and monovalent cations, including but not limited to Na+. + Mg 2+ and K + .

[0080] In some embodiments, the sample is maintained at a temperature that preserves the viability of any pathogen cells present in the sample. During the step in which the phage attaches to bacterial cells, the sample may be maintained at a temperature that promotes phage activity. Such a temperature is at least about 25°C and does not exceed 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 phage binding or infection.

[0081] Embodiments of the present invention can utilize various suitable control samples. For example, control samples that do not contain bacteriophages or control samples that contain bacteriophages but do not contain the microorganism of interest can be used as controls for determining background signal levels.

[0082] Indicator phages with reproductive defects

[0083] As described in more detail herein, compositions, methods, systems, and kits according to embodiments of the present invention may include indicator phages for detecting reproductive defects in pathogenic microorganisms. In some embodiments, the present invention includes recombinant reproductive defect indicator phages having one or more gene modifications to include an indicator gene and such that the phage is reproductively defective. The aforementioned gene modifications may be introduced during one gene modification step or during multiple gene modification steps (such as two or more gene modification steps). In some embodiments, the present invention may include compositions comprising reproductive defect indicator phages.

[0084] Recombinant reproductively deficient indicator phages may include reporter genes or indicator genes. In some embodiments of the infection source, the indicator gene does not encode a fusion protein. For example, in some embodiments, expression of the indicator gene after infection of a host microorganism (such as bacteria) produces a soluble indicator protein product. In some embodiments, the indicator gene may be inserted into the late gene region of the reproductively deficient indicator phage. Because late genes encode structural proteins, they are generally expressed at higher levels than other phage genes.

[0085] Recombinant reproductively deficient indicator phages according to embodiments of the present invention contain alterations that prevent the recombinant phage from replicating after infecting a host organism. Suitable genes and alterations are selected based on several considerations. Suitable phage genes for alteration are those that affect the ability of the phage to replicate in the host microorganism after infection but do not affect the ability of the recombinant reproductively deficient phage to infect the host microorganism. In some embodiments, genes altered to make the recombinant phage reproductively deficient are selected such that they are not genes required for phage genome replication. This ensures that the recombinant phage genome replicates at a typically high copy number, producing a high copy number of indicator genes. Early and immediate early genes are often classified as genes required for phage genome replication. Early and immediate early genes (e.g., T7 RNA polymerase) can also be required for gene expression controlled by late gene promoters, such as indicator genes in recombinant phages. Therefore, immediate early and early genes, also referred to as class I or class II genes, may not be suitable for alteration. In some embodiments, genes altered to make the recombinant phage reproductively deficient are selected because they are required for the production of mature phage viral particles. For example, suitable genes for alteration or deletion can be structurally important genes, such as those required for viral particle assembly. In some embodiments, genes selected to be altered to make the recombinant phage reproductively deficient are late-stage genes required for the production of mature phage viral particles and not expressed at high copy numbers. In some embodiments, a reproductively deficient indicator phage may contain more than one (i.e., one or more) altered genes. Some examples of genes that could be suitable for alteration or deletion to make recombinant phages reproductively defective are as follows: In phage T4 and related phages, as well as T4 viruses (e.g., SEA1, Saka4, and TSP12 phages) and closely related Viulikeviruses (e.g., CBA120, TSP1 phages), some of the genes suitable for alteration are: gp4 encoding the head completion protein; gp20 encoding the portal vertex protein; gp21 encoding the front head core scaffold protein and protease; gp22 encoding the front head scaffold protein; gp25 encoding the substrate wedge subunit; gp26 encoding the substrate apical subunit; gp53 encoding the substrate wedge assembly; and gp54 encoding the substrate-tail tube initiation protein. In short-tailed viruses (T7, MP87 phages), some of the genes that can be modified are: gp6.7 encoding viral particle proteins; gp7.3 encoding tail proteins; gp8 encoding head-tail connector proteins; gp9 encoding scaffold proteins; and gp13.In long-tailed viruses (T5, P70-associated phages), some of the genes suitable for modification include Gp150, which encodes the protoprotease, and gp152, which encodes the entry protein. It should be understood that the above list is non-restrictive, and other genes can be modified in a wide variety of phages.

[0086] In some embodiments, the reproductively deficient indicator phage according to embodiments of the invention contains a mutation in a suitable gene. Such mutations can be amber mutations, ochre mutations, base substitutions, deletions or insertions, or any combination of the above types. Mutations or combinations thereof can render the selected gene nonfunctional by altering the structure of the encoded protein, suppress transcription or expression of the altered gene (e.g., by changing the promoter), leading to premature termination of transcription or expression, etc. In some other embodiments, in the reproductively deficient indicator phage, the suitable gene is altered 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, it is preferable to replace at least a portion of the suitable gene in the phage with an indicator gene rather than introducing one or more mutations into the suitable gene, thereby avoiding the reversion or suppression of said one or more mutations in the suitable gene and the recombinant phage reverting to a reproductively competent state.

[0087] In some implementations, the reproductive defect indicator phage may be derived from short-tailed viruses (such as T7, T7-like), myotailed viruses (such as T4, T4-like), ViI, ViI-like (or Vi1 virus, according to GenBank / NCBI), Cronobacter spp.-specific phages (such as Saka2 or Saka4), Salmonella spp. phage SPN1S, Salmonella spp. phage 10, Salmonella spp. phage ε15, Salmonella spp. phage SEA1, Salmonella spp. phage Spn1s, Salmonella spp. phage P22, Salmonella spp. phage TSP1, Salmonella spp. phage TSP11, or Listeria spp. Phages LipZ5, Listeria spp. phage P40, Listeria spp. phage vB_LmoM_AG20, Listeria spp. phage P70, Listeria spp. phage A511, Listeria spp. phage LMA4, Listeria spp. phage LMA8, Listeria spp. phage LPES1, Listeria spp. phage LPJP1, Staphylococcus spp. phage P4W, Staphylococcus spp. phage K, Staphylococcus spp. phage Twort, Staphylococcus spp. phage SA97, Staphylococcus spp. phage ISP, Escherichia coli O157:H7 phage CBA120, or other wild-type or engineered phages. In some implementations, the indicator phage is derived from a phage whose genome has 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% homology with phages derived from the following: short-tailed viruses, such as T7, T7-like; myotailed viruses, such as T4, T4-like; ViI, ViI-like (or Vi1 virus, according to GenBank / NCBI); Kronobacter spp. specific phages, such as Saka2 or Saka4; Salmonella spp. phages. The phages used include SPN1S, Salmonella spp. phage 10, Salmonella spp. phage ε15, Salmonella spp. phage SEA1, Salmonella spp. phage Spn1s, Salmonella spp. phage P22, Listeria spp. phage LipZ5, Listeria spp. phage P40, Listeria spp. phage vB_LmoM_AG20, Listeria spp. phage P70, Listeria spp. phage A511, Staphylococcus spp. phage P4W, Staphylococcus spp. phage K, Staphylococcus spp. phage Twort, Staphylococcus spp. phage SA97, Escherichia coli O157:H7 phage CBA120, or other wild-type or engineered phages. In some embodiments, the indicator phage for reproductive defects is derived from a phage highly specific to the specific microorganism. For example, reproductive defect indicator phages can be prepared from environment-derived phages specific to bacteria found in certain environments.

[0088] The selection of indicator genes to be inserted into indicator phages with reproductive defects can be guided by a variety of considerations. For example, most phages can package DNA that is several percent larger than their natural genome. Under such considerations, smaller indicator genes may be a more suitable choice for modifying phages (especially those with smaller genomes). OpLuc and The protein is only about 20 kDa (encoding approximately 500-600 bp), while FLuc is about 62 kDa (encoding approximately 1,700 bp). For comparison, the T7 genome is approximately 40 kbp, and the T4 genome is approximately 170 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacteria (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily and timely detected. of It is a modified luciferase from *Oplophorus gracilirostris* (deep-sea shrimp). In some implementations, With NanoGlo (also) The product, a combination of imidazopyrazinone substrate (furimazine), can provide a stable signal with low background. In some embodiments, more than one indicator gene can be inserted into the reproduction-deficient phage. For example, more than one copy (such as two copies) of the same indicator gene can be inserted, which can improve the signal intensity and / or signal-to-noise ratio of assays using reproduction-deficient indicator phages. In another embodiment, different indicator genes, such as two different indicator genes, can be inserted, which can allow for bimodal signal detection. For example, The gene can be inserted together with the gene encoding green fluorescent protein (GFP), or The gene can be inserted along with genes encoding different luciferases (such as firefly luciferase).

[0089] Indicator genes can encode a wide variety of biomolecules or can themselves be detectable biomolecules. For example, an indicator gene can encode a detectable polypeptide or protein. In another embodiment, an indicator gene can be a gene expressing a detectable product or a gene encoding an enzyme that produces a detectable product. In yet another example, an indicator gene can encode or include a detectable nucleic acid. For example, an indicator gene can encode a detectable aptamer, such as RNA mango, or an indicator gene can contain a nucleic acid sequence detectable using real-time polymerase chain reaction (RT-PCR). In some embodiments, the product of an indicator gene can be a detectable enzyme. The indicator gene product can generate light and / or can be detected 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 serve as the indicator portion. For example, in some embodiments, the indicator gene encodes luciferase. Various types of luciferase can be used. The luciferase can be one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renida luciferase, or engineered luciferase. In some embodiments, firefly luciferase is the indicator part. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as... Other engineered luciferases or other enzymes that generate detectable signals can also be suitable indicator components.

[0090] Genetic modifications to reproductively defective indicator phages may include the insertion, deletion, or substitution of small nucleic acid fragments, a large portion of the gene, or the entire gene. In some embodiments, the inserted or substituted nucleic acid contains a non-natural sequence. For example, a non-natural indicator gene may be inserted into the phage genome such that it is under the control of a phage promoter. A non-natural indicator gene may be inserted such that it replaces at least a portion of the sequence of a late-stage phage gene, and the insertion of the indicator gene makes the resulting recombinant phage reproductively defective. Including a stop codon in all three reading frames of the indicator gene can help increase expression by reducing readthrough, also known as leaky expression. This strategy can also eliminate the possibility of fusion proteins being prepared at low levels, which would manifest as a background signal that cannot be separated from the phage. Therefore, in some embodiments, the non-natural indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification may be configured such that the indicator protein product does not contain a phage polypeptide. In some embodiments, the present invention includes genetically modified reproductively defective indicator phages containing a non-phage indicator gene in a late (class III) gene region. In some embodiments, the non-natural indicator gene is under the control of a late-stage promoter. Using a late viral gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like a viral capsid protein, but is not shut down as in similar endogenous bacterial genes or early phage genes. In some implementations, the late promoter is a T4-, T7-, or ViI-like promoter, or another phage promoter similar to those found in wild-type phages.

[0091] In some embodiments, expression of the indicator gene of the reproduction-deficient indicator phage in the microorganism of interest, following infection with the phage, leads to the production of a soluble protein product. In some embodiments, the non-natural indicator gene is not adjacent to the gene encoding the structural phage protein, thus no fusion protein is produced. Unlike systems employing detection of fusions (fusion proteins) between the detection portion and the capsid 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 contain the phage structure. That is, the indicator or reporter is not attached to the phage structure. Thus, the gene of the indicator or reporter does not fuse with other genes in the genome of the reproduction-deficient indicator phage. This can greatly increase the sensitivity of the detection assay using the reproduction-deficient indicator phage according to embodiments of the present invention (sensitivity can be increased down to detecting a single microorganism in the sample) and simplify the assay, such that for some embodiments the assay is completed in 2 hours or less, unlike the several hours required by the additional purification steps required to produce a construct of a detectable fusion protein. Furthermore, fusion proteins may have lower activity than soluble proteins, for example, due to protein folding constraints, which can alter the conformation of the enzyme's active site or its proximity to the substrate. If the concentration is 10 bacterial cells / mL sample, for example, for the assay described above, less than 2 hours may be sufficient.

[0092] Furthermore, by definition, fusion proteins limit the number of portions that attach to protein subunits in the phage. For example, a commercially available system using a platform designed to act as a fusion protein would produce approximately 415 copies of the fusion portion, corresponding to approximately 415 copies of the gene 10B capsid protein in each T7 phage particle. Without this constraint, infected bacteria are expected to express far more copies of detection portions (e.g., luciferase) than can fit on the phage.

[0093] In some embodiments of recombinant reproductively deficient indicator phages, late promoters (such as class III promoters, e.g., from T7, T4, ViI, or Saka) are used for transcription of indicator genes. These late promoters have high affinity for the RNA polymerase of the same phage that transcribes the genes of structural proteins assembled into the phage particle. These proteins are among the most abundant proteins produced by phages, as each phage particle contains tens or hundreds of copies of these molecules. Using viral late promoters ensures optimal high levels of expression of indicator gene products (such as luciferase). Using late promoters from the original wild-type phage derived from the reproductively deficient indicator phage, late promoters specific to the original wild-type phage, or late promoters active under the control of the original wild-type phage (e.g., T4, T7, ViI, or Saka late promoters and T4-, T7-, ViI-, or Saka-based systems), further ensures optimal expression of the detection portion. In some cases, using standard bacterial (non-viral / non-phage) promoters may be detrimental to expression because these promoters are often downregulated during phage infection (so that phages prioritize bacterial resources for phage protein production). Therefore, in some embodiments, reproductively deficient indicator phages are preferably engineered to encode and express soluble (free) indicator moieties at high levels, said engineering being performed using locations in the genome that do not restrict expression to the number of subunits of phage structural components.

[0094] In some embodiments, reproductively deficient indicator phages are designed to optimize desired traits for use in assays detecting microorganisms of interest. In some embodiments, bioinformatics and prior analysis of gene modifications are employed to optimize the desired traits. For example, in some embodiments, genes encoding phage tail proteins may be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins may be optimized to recognize and bind to bacteria of an entire genus, or species of a specific group within that genus. In this way, phages can be optimized to detect a broader or narrower group of pathogens. In some embodiments, reproductively deficient indicator phages may be designed to increase the expression of reporter genes. Additionally and / or alternatively, in some cases, reproductively deficient indicator phages may be designed to increase phage lysis rates to improve detection. Designing reproductively deficient indicator phages to produce increased copy numbers of the phage genome post-infection or to increase the expression levels of late-stage genes will result in increased lysis rates.

[0095] In some embodiments, the stability of reproductively deficient indicator phages can be optimized to improve shelf life. For example, enzyme-antibiotic solubility can be increased to enhance subsequent phage stability. Additionally and / or alternatively, the thermal stability of reproductively deficient indicator phages can be optimized. Thermostable phages retain better functional activity during storage, thereby increasing shelf life. Therefore, in some embodiments, thermal stability and / or pH tolerance can be optimized.

[0096] The compositions of the present invention may comprise one or more reproduction-deficient indicator phages and one or more indicator genes. In some embodiments, the composition may comprise a mixture of different reproduction-deficient indicator phages specific to different microorganisms of interest. Such mixtures can be used to simultaneously detect multiple microorganisms of interest. In some embodiments, the composition may comprise a mixture of different reproduction-deficient indicator phages that can encode and express the same or different indicator proteins. In some embodiments, the mixture of reproduction-deficient phages comprises at least two different types of reproduction-deficient indicator phages.

[0097] Methods for preparing (creating) reproductively defective indicator phages

[0098] Implementations of methods for preparing reproductively deficient indicator phages may begin with the selection of parental phages for genetic modification. For example, some phages are highly specific to target microorganisms, which may include specificity for a particular strain or serotype of the target microorganism. This presents opportunities for highly specific detection. The parental phage can be a wild-type phage found in any environment or an engineered phage. Methods according to embodiments of the invention utilize the high binding specificity associated with the phage, which recognizes and binds to a specific microorganism of interest as an amplified signal, thereby detecting low levels of microorganisms (in some cases, down to a single microorganism) present in a sample. For example, the phage specifically recognizes surface receptors of specific microorganisms and thus specifically infects those microorganisms. Thus, they are suitable for targeting microorganisms of interest. Some embodiments of the invention utilize the binding specificity and high-level gene expression capacity of indicator phages for rapid and sensitive targeting to infect and facilitate the detection of microorganisms of interest. Therefore, some embodiments of methods for preparing recombinant reproductively deficient indicator phages may include steps related to selecting parental phages that specifically infect target microorganisms of interest.

[0099] Some embodiments of methods for preparing recombinant reproductively deficient indicator phages include the step of altering one or more genes of a parental phage to produce a recombinant reproductively deficient phage. For example, some embodiments may include the step of introducing one or more mutations into suitable genes to make the parental phage reproductively deficient. Such suitable genes and mutations are described elsewhere in this document.

[0100] Some embodiments of a method for preparing recombinant reproductively deficient indicator phages include the step of altering the genes of a parental phage to produce a recombinant reproductively deficient phage. For reproduction, a reproductively deficient phage requires an engineered strain of the host microorganism (such as bacteria) capable of expressing a gene that has been altered to make the phage a product of a reproductively deficient gene. Such engineered strains may be referred to as "permissive." Therefore, some embodiments of a method for preparing recombinant reproductively deficient indicator phages may include the step of generating such permissive engineered strains of the host microorganism. Some embodiments of a method for preparing recombinant reproductively deficient indicator phages may include the step of infecting a permissive engineered strain of the host microorganism with the reproductively deficient indicator phage. Some embodiments of a method for preparing recombinant reproductively deficient indicator phages may include the step of preparing a homologous recombinant plasmid / vector containing the indicator gene. Some embodiments of a method for preparing recombinant reproductively deficient indicator phages may include the step of transforming the homologous recombinant plasmid / vector into a permissive engineered host microorganism infected with the reproductively deficient indicator phage. Other embodiments of the method for preparing recombinant reproductively deficient indicator phages may include the steps of transforming the homologous recombinant plasmid / vector into a permissible engineered host microorganism, followed by infecting the transformed permissible engineered host microorganism with the reproductively deficient indicator phage. In some embodiments, infection of the permissible engineered host microorganism and transformation of the homologous recombinant plasmid / vector into the permissible engineered host microorganism may be performed in the same step. In other embodiments, infection of the permissible engineered host microorganism and transformation of the homologous recombinant plasmid / vector into the permissible engineered host microorganism may be performed in two or more steps. Once the permissible engineered host microorganism accepts the reproductively deficient phage and the homologous recombinant plasmid / vector, homologous recombination occurs between the plasmid / vector and the phage genome. The recombinant reproductively deficient phage containing the indicator gene (recombinant reproductively deficient indicator phage) can then be isolated.

[0101] In some embodiments, altering the parental phage to make it reproductively defective can be achieved by replacing at least a portion of the parental phage with an indicator gene. Therefore, some embodiments of a method for preparing recombinant reproductively defective indicator phages include the step of preparing a homologous recombinant plasmid / vector containing an indicator gene flanked by a sequence of a targeted gene that is deleted in the parental phage to make it reproductively defective. The homologous recombinant plasmid / vector can then be transformed into a permissible engineered host microorganism infected with the parental phage, thereby allowing homologous recombination between the plasmid / vector and the parental phage genome. Some other embodiments of a method for preparing recombinant reproductively defective indicator phages may include the step of transforming the homologous recombinant plasmid / vector into a permissible engineered host microorganism, followed by infecting the transformed permissible engineered host microorganism with the reproductively defective indicator phage, thereby allowing homologous recombination between the plasmid / vector and the parental phage genome. In some implementations, infection of the permissible engineered host microorganism and transformation of the homologous recombination plasmid / vector into the permissible engineered host organism can be achieved in the same step, thereby allowing homologous recombination to occur between the plasmid / vector and the parental phage genome. The reproductively deficient phage containing the indicator gene can then be isolated (reproductively deficient indicator phage).

[0102] In some embodiments of the method for preparing recombinant reproductively deficient indicator phages, the introduction of reproductively deficient and indicator genes into the parental phage is achieved in a one-step recombination process. A recombination strategy for such a process is illustrated in... Figure 1 The advantages of such implementation schemes include simplifying the process of generating reproductively deficient indicator phages. Another advantage of these implementation schemes is that they allow the use of reporter genes to detect and isolate reproductively deficient indicator phages. If genetic alterations are introduced to confer reproductive deficiencies on reproductively deficient phages that include the indicator gene (reproductive indicator phages), both phages with and without the reproductively deficient alterations will grow, making the screening of indicator phages more difficult.

[0103] Figure 3 The diagram schematically illustrates a homologous recombination process occurring between a host microorganism's homologous recombination (HR) plasmid and a parental bacteriophage genome, resulting in the generation of an indicator bacteriophage according to one embodiment of the invention. In the illustrated homologous recombination process, the bacteriophage is CBA120 *Escherichia coli* bacteriophage, and the reporter gene is... Reporter gene. Therefore, in the illustrated implementation, the host microorganism is *Escherichia coli*. It is to be understood that... Figure 3This is intended to be exemplary and non-limiting; other phages, corresponding host organisms, and reporter genes may be used. In some embodiments, phages isolated from the environment may be preferably used to prepare reproductively defective indicator phages. In this way, reproductively defective indicator phages specific to microorganisms of natural origin can be generated.

[0104] Various methods for designing and preparing homologous recombination plasmids are known. Various methods for transforming bacteria with plasmids are known, including heat shock, F-ciliary-mediated bacterial conjugation, electroporation, and others. Various methods for isolating specific clones after homologous recombination are also known. Some of the methods described herein utilize specific strategies.

[0105] Some embodiments of a method for preparing reproductively deficient indicator phages may include the steps of selecting a parental phage specifically to infect a target microorganism of interest; determining the native sequence in the late region of the selected parental phage; annotating the genome and identifying a suitable late gene of the selected parental phage, wherein the suitable late gene is modified to make the parental phage reproductively deficient; designing a sequence adjacent to the major late gene for homologous recombination and including a codon-optimized reporter gene; inserting the sequence designed for homologous recombination into a plasmid / vector; transforming the plasmid / vector into a target microorganism containing a plasmid encoding a suitable late gene that functions; selecting the transformed target microorganism; infecting the transformed microorganism with the selected parental 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 limiting dilution assays to enrich and isolate the recombinant phage. Enrichment of reproductively deficient recombinant phage fractions in parental phage fractions may be performed wholly or partially in permissible target cells containing a trans plasmid. Some embodiments include repeating the limiting dilution and titration steps as needed after the initial limiting dilution assay until the recombinant phage represents a detectable fraction of the mixture. For example, in some embodiments, the limiting dilution and titration steps may be repeated until at least 1 / 30 of the phage in the mixture is recombinant before isolating a specific clone of the recombinant phage. In some embodiments, a 1:30 recombinant:parent ratio is expected to produce an average of 3.2 transduction units (TUs) per 96 plaques (e.g., in a 96-well plate). The initial ratio of recombinant to parental phage can be determined by limiting dilution assays based on TCID50 (50% of the tissue culture infectious dose) as previously described in U.S. Application No. 15 / 409,258. By Poisson distribution, the probability of observing at least one TU somewhere in a 96-well plate produced by a 1:30 ratio is 96%.

[0106] Some embodiments involve designing (and optionally preparing) a sequence for homologous recombination that requires the insertion of an indicator gene. In some embodiments, the homologous recombination sequence is designed to replace the late gene to make the parental phage reproductively defective. In some embodiments, the indicator gene sequence comprises a codon-optimized reporter gene preceding an untranslated region. The untranslated region may include a phage late gene promoter and a ribosome entry site. In some embodiments, the inserted gene construct further includes its own exogenous dedicated promoter to drive the expression of the indicator gene. An exogenous promoter is a promoter other than any endogenous promoter in the phage genome. When a phage produces a polycistronic mRNA transcript, only a single promoter is required upstream of the first gene / cistron of the transcript. Conventional recombination constructs use only endogenous phage promoters to drive the inserted gene. Adding an additional promoter upstream of the reporter gene and ribosome binding site can increase gene expression by acting as a second initiation site for transcription.

[0107] Numerous known methods and commercially available products are available for plasmid preparation. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be combined to prepare plasmids. Synthetic plasmids are also commercially available (e.g., GeneWiz). Glusomes can also be used, or the CRISPR / CAS9 system can be used to selectively edit the phage genome. Some embodiments of methods for preparing indicator phages involve designing plasmids that can be readily recombinated with the starting phage genome to generate a recombinant genome. In designing plasmids, some embodiments include adding codon-optimized reporter genes, such as luciferase genes. Some embodiments further include adding elements upstream of the untranslated region. For example, an upstream untranslated region can be added to the coding region. The untranslated region precedes the start codon sequence of the reporter gene. The untranslated region may include promoters such as T4, T4-like, T7, T7-like, ViI, or ViI-like promoters. The untranslated region may also include ribosome entry / binding sites (RBS), also known as the “Shine-Dalgarno sequence” of the bacterial system. Any one or two of these elements, or other untranslated elements, may be embedded within a short upstream untranslated region consisting of a random sequence containing approximately the same GC content as the rest of the phage genome. The random region should not include the ATG sequence, as the ATG sequence will serve as the start codon.

[0108] As discussed elsewhere in this document, the isolation and propagation of recombinant reproductively deficient indicator phages according to embodiments of the invention can be carried out only in “permissible” host microorganisms that express one or more genes that have been altered to make the reproductively deficient indicator phage reproductively deficient. Such “permissible” microorganisms, such as bacteria, can be modified by transforming them with plasmids expressing genes required for phage propagation. Figure 2 The illustration shows cells transformed with plasmids expressing genes required for phage reproduction and infected with a reproductively defective indicator phage. Plasmids containing genes required for phage reproduction are selected to be compatible with homologous recombinant plasmids used to insert the indicator genes into the phage genome. For example, plasmids expressing genes required for phage reproduction and homologous recombinant plasmids can be selected such that they contain different antibiotic resistance markers, allowing both plasmids to remain in the host organism simultaneously. In another embodiment, plasmids expressing genes required for phage reproduction and homologous recombinant plasmids are selected to contain compatible replication origins so as not to interfere with each other. Examples of compatible plasmids are pUC-derived plasmids using the ori replication origin and pBAV1k-T5-GFP plasmids using the RCR (rolling circular replication) replication origin. Because reproductively deficient phages require engineered “allowable” strains of the host microorganism to replicate, some embodiments of methods for preparing recombinant reproductively deficient indicator phages include one or more steps of generating an engineered strain of the target microorganism capable of expressing the product of genes in the reproductively deficient phage that have been altered to make it reproductively deficient. In some embodiments, generating the engineered strain of the target microorganism involves transforming the target microorganism with a plasmid encoding one or more genes that are altered in the recombinant reproductively deficient phage. Alternatively, the desired gene can be integrated into the target microorganism genome by various other methods, such as via transposons, homologous recombination, site-directed recombination / integration, or other methods.

[0109] Figure 27An example of a process for isolating reproductively deficient recombinant phages from a mixture of parental phages and reproductively deficient indicator phages derived from homologous recombination is illustrated. In step 402, a permissible host microorganism transformed with a homologous recombination plasmid and a plasmid expressing the desired phage gene is infected with the parental phage, yielding progeny phages 434 containing a mixture of the parental and reproductively deficient recombinant indicator phages, wherein the ratio of parental phages to reproductively deficient indicator phages is very low. The resulting phage mixture is diluted 404 into 96-well plates 406, yielding an average of 5 recombinant transduction units (TUs) / plate (9.3 PFU / well). As described below, the reporter gene activity of the 96-well plates is then measured to identify wells 436 containing reproductively deficient indicator phages, compared with wells 440 containing parental phages. A permissible host microorganism 438 containing a plasmid expressing the desired phage gene is added to each well (408); for example, when the host microorganism is bacteria, each well may contain approximately 50 μL of turbid bacterial culture. This allows the reproductively deficient indicator phage to replicate and produce a soluble reporter gene product 442. After incubation step 410 (e.g., incubation at 37°C for 5 hours), the presence of reporter gene product 442 in the wells can be screened. Any positive well may have been inoculated with a single reproductively deficient indicator phage, and at this stage, the mixture may contain an enrichment of approximately 10 parental phages:1 recombinant, exceeding the original ratio. If desired (e.g., if the recombinant:to-total ratio is less than 1:30), progeny 412 from this enriched culture may be subjected to one or more additional limiting dilution assays 414 to increase the ratio and determine the actual concentration of recombinant reproductively deficient indicator phage transduction units. For example, if the ratio is 1:384 recombinant:PFU (where PFU is determined by plaque assay on permissible bacteria), then approximately 5 recombinant TUs and 1920 contaminated phages (5 x 384 = 1920) per well of a 96-well plate 416 can be ablated from the previous positive wells 414 to obtain an approximate inoculum of 20 predominantly parental phages per well of a second dilution assay plate 420 (1920 PFU / 96 wells = 20 PFU / well). Any positive luciferase well may be inoculated with a single recombinant reproductively deficient indicator phage and 19 parental phages. These wells can be analyzed for the presence of luciferase 442.

[0110] After adding the host microorganism and incubating for 418, the soluble reporter gene product and phage are present at approximately a 20:1 ratio 420. This ratio can be verified by TU50 titration of the recombinant and plaque assay of total PFU. TU50 titration is a limiting dilution assay based on tissue culture infectious dose 50 (TCID50) to score reporter gene product activity rather than cell killing. Finally, plaque assay 422 can be performed to screen 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 436 in a third multi-well plate 426. In some embodiments, this should ensure that enough plaques are screened such that approximately three indicator phages are present in the mixture of screened plaques based on a known ratio of recombinant to total phage. A plaque can be removed from the plate to each well of a 96-well plate 424, and reporter gene product assay 426 can be performed to determine which well contains phages exhibiting reporter gene product activity 442. Well 428, demonstrating such activity, represents pure recombinant reproductively deficient indicator phage 434, while well 430, lacking such activity, represents pure parental phage 432. Individual plaques can then be suspended in buffer (e.g., 100 μL buffer) or culture medium, and aliquots (e.g., about 5 μL) can be added to wells containing a culture of the host microorganism, followed by assaying after incubation (e.g., at 37°C for about 45 minutes to 1 hour). Positive wells are expected to contain a pure culture of the reproductively deficient indicator phage. Some embodiments may include additional rounds of plaque purification. Thus, as... Figure 27 As shown, reproductively deficient indicator phages produced by homologous recombination of plasmids designed for recombination with the parental phage genome can be isolated from a mixture containing a very small percentage (e.g., 0.005%) of indicator phages.

[0111] Following separation, large-scale production can be carried out to obtain a high-titer indicator phage stock solution suitable for use in detection methods according to embodiments of the invention. The production and preparation of the indicator phage stock solution may include purifying the indicator phage from any free detection fraction generated during the production of the indicator phage in bacterial cultures. Standard phage purification techniques can be used to purify some embodiments of the phage according to the invention, such as sucrose density gradient centrifugation, cesium chloride isodensity gradient centrifugation, HPLC, size exclusion chromatography, and dialysis or derivatization techniques (such as Amicon concentrators – Millipore, Inc.). As a result of the purification process, the indicator phage stock solution may be substantially free of any reporter gene products generated during production. Removing residual indicator gene products present in the indicator phage stock solution can significantly reduce the background signal observed when the indicator phage is used to detect microorganisms of interest in a sample.

[0112] [Methods for detecting microorganisms using indicator phages with reproductive defects]

[0113] As described herein, in some embodiments, the present invention may include a method for detecting microorganisms using a reproduction-deficient indicator phage. The method for detecting microorganisms using a reproduction-deficient indicator phage according to embodiments of the present invention can be implemented in a wide variety of ways.

[0114] In one embodiment, the present invention may include a method for detecting a microorganism of interest in a sample, comprising the steps of: incubating the sample with a reproductively deficient indicator phage infecting the microorganism of interest, wherein the reproductively deficient indicator phage contains an indicator gene such that expression of the indicator gene after infection of the microorganism of interest results in the production of an indicator gene product; and detecting the indicator gene product, wherein a 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 present invention may include a method for detecting a microorganism of interest in a sample, comprising the steps of: incubating the sample with a reproductively deficient indicator phage infecting the microorganism of interest, wherein the reproductively deficient indicator phage contains an indicator gene such that expression of the indicator gene after infection of the microorganism of interest results in the 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 the soluble indicator gene product) indicates that the microorganism of interest is present in the sample. In another embodiment, the present invention may include a method for detecting a microorganism of interest in a sample, comprising the steps of: incubating the sample with a reproductively deficient indicator phage infecting the microorganism of interest, wherein the reproductively deficient indicator phage contains an indicator gene such that expression of the indicator gene after infection of the microorganism of interest results in the 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 the soluble indicator gene product protein) indicates that the microorganism of interest is present in the sample. In variations of the above embodiments, the microorganism of interest may be a bacterium of interest. For example, in an exemplary embodiment, the present invention may include a method for detecting bacteria of interest in a sample, comprising the steps of: incubating the sample with a reproductively deficient indicator phage infecting the bacteria of interest, wherein the reproductively deficient indicator phage contains an indicator gene such that expression of the indicator gene after infection of the bacteria of interest results in the production of a soluble indicator gene product; and detecting the indicator gene product, wherein a positive detection of the indicator gene product indicates that the bacteria of interest are present in the sample.

[0115] In some embodiments, methods for detecting microorganisms of interest using reproductively deficient indicator phages (such methods may be referred to as "assays") can be performed to utilize a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments employing the reproductively deficient indicator phages (i.e., indicator phages) of the present invention can allow for rapid detection of specific bacterial strains, depending on sample type, sample size, and assay format, with total assay times of less than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5. 11.0, 11.5, 12, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, or 26.0 hours. For example, the required time can be shorter or longer depending on the phage strain and the bacterial strain to be detected in the assay, the type and size of the sample to be tested, the conditions required for target activity, the complexity of the physical / chemical environment, and the concentration of “endogenous” non-target bacterial contaminants.

[0116] Figure 28 This illustrates a strategy based on an embodiment of the invention using a reproductively deficient indicator phage that produces soluble luciferase. In this method, the reproductively deficient indicator phage can be modified to express soluble luciferase. Luciferase expression is driven by a viral capsid promoter (e.g., a late T7 or T4 promoter from phage), resulting in high expression. Figure 28In the embodiments shown, at least a portion of a sample 500 containing the microorganism 502 to be detected is placed in a rotary column filter and centrifuged to remove excess liquid. A suitable plurality of reproductively deficient indicator phages 504, genetically modified to express soluble luciferase 503, are added. Infected cells may be incubated for a time sufficient for infection to occur (e.g., at 37°C for 30–240 minutes). In some embodiments, cell lysis may occur. In other embodiments, cells may not lyse. The reproductively deficient indicator phages 504 plus free luciferase 503 in the lysate can then be collected, for example, by centrifugation, and the luciferase level in the filtrate can be quantified using a spectrophotometer 518. Alternatively, a high-throughput method can be used, wherein the sample is applied to a 96-well plate, and after performing all the operations listed above, the luciferase in the original 96-well plate can be directly measured without a final centrifugation step. Other simplified or self-prepared methods may also be used, as previously described. After infection with a reproductively defective indicator phage, such methods may not require centrifugation or further separation of any components. In some embodiments, a single device with 2, 3, 4 or more chambers can be used to perform the assay infection and incubation steps, followed by detection using a suitable device, such as a handheld photometer to detect luminescence.

[0117] Figure 29 A plate assay for detecting microorganisms of interest using a reproductively deficient indicator phage according to an embodiment of the invention is described. Briefly, a sample 616 containing the microorganism of interest 618 can be added to the wells 602 of a porous filter plate 604 and centrifuged 606 to concentrate the sample by removing liquid. A reproductively deficient indicator phage 620 is added to the wells and incubated with additional culture medium for a sufficient time 608 for adsorption, followed by infection of the target microorganism of interest and advancement of the phage life cycle 610 (e.g., ~45 min–2 h) to enable late-stage gene production by the reproductively deficient indicator phage, which typically occurs late in the infection cycle (but the reproductively deficient indicator phage does not produce any mature viral particles). Finally, a luciferase substrate is added and reacted with any present luciferase 624. The generated emission is measured in a photometer 614, detecting luciferase activity 626.

[0118] In some embodiments, the assay can be performed on or near the captured surface without concentrating the microorganisms of interest. Figure 30The illustration depicts a “concentration-free assay” for detecting microorganisms of interest using a reproductively deficient indicator phage according to an embodiment of the invention. Aliquots of the reproductively deficient indicator phage 714 are dispensed into individual wells 702 of a multi-well plate 704, followed by the addition of aliquots of the test sample containing the microorganism of interest 712 and incubation 706 (e.g., at 37°C for 45 minutes) for a period sufficient to allow the phage to generate a soluble indicator 716 (e.g., luciferase). The plate wells 708 containing the soluble indicator and the reproductively deficient indicator phage can then be measured to determine the indicator activity (e.g., luciferase assay) on plate 718. In this embodiment, the test sample is not concentrated (e.g., by centrifugation) but is simply incubated directly with the reproductively deficient indicator phage for a specified period, followed by measurement of luciferase activity.

[0119] In some implementations, samples can be enriched by incubation under growth-promoting conditions prior to testing. In such implementations, depending on the sample type and size, 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.

[0120] In some embodiments, the reproductively deficient indicator phage includes a detectable indicator portion, and infection of a single pathogenic cell (e.g., bacteria) can be detected by an amplified signal generated via the indicator portion. Therefore, the method may include detecting the indicator portion generated during infection with the reproductively deficient indicator phage, wherein detection of the indicator indicates the presence of a microorganism of interest (such as bacteria of interest) in the sample. In an exemplary embodiment, the invention may include a method for detecting bacteria of interest in a sample, comprising the steps of: incubating the sample with a reproductively deficient indicator phage infecting the bacteria of interest, wherein the reproductively deficient indicator phage includes an indicator gene inserted into a late gene region of the phage such that expression of the indicator gene after infection with the bacteria of interest results in the production of a soluble indicator gene product; and detecting the indicator gene product, wherein a positive detection of the indicator gene product (i.e., a detection of presence, level, or amount) indicates the presence of the bacteria of interest in the sample. In some embodiments, the amount of the detected indicator portion corresponds to the amount of bacteria of interest present in the sample.

[0121] As described in more detail herein, the methods and systems according to embodiments of the invention can utilize a range of concentrations of reproductively deficient indicator phages to infect microorganisms of interest (such as bacteria) that may be present in a sample. In some embodiments, reproductively deficient indicator phages are added to the sample at concentrations sufficient to rapidly detect, bind to, and infect target microorganisms (such as bacteria) present in very low numbers (such as single cells). In some embodiments, the concentration of reproductively deficient indicator phages may be sufficient to detect, bind to, and infect target bacteria in less than 1 hour. In other embodiments, these events may occur in less than 2 hours or less than 3 hours after the reproductively deficient indicator phages are added to the sample. For example, in some embodiments, the concentration of reproductively deficient indicator phages used for the incubation step is greater than 1 x 10⁻⁶. 5 PFU / mL, greater than 1 x 10 6 PFU / mL, greater than 1 x 10 7 PFU / mL, or greater than 1 x 10 8 PFU / mL.

[0122] In some embodiments of a method for detecting microorganisms of interest in a sample, the reproduction-deficient indicator phage may be purified to remove any residual indicator proteins that may have been generated during the production of the infectious source stock solution, prior to the step of incubating the sample with a reproduction-deficient indicator phage infecting the microorganism of interest. Therefore, in some embodiments, the method may include the step of purifying the reproduction-deficient indicator phage. Recombinant reproduction-deficient indicator phages can be purified by various methods, such as by centrifugation using a cesium chloride isodense gradient prior to incubation with the sample. Purification may have the additional benefit of removing phages that do not possess DNA (i.e., empty phages or “ghosts”).

[0123] In some embodiments of the method of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in some embodiments, a sample containing one or more microorganisms of interest can be directly applied to an assay container such as a centrifuge column, microtiter well, or filter, and the assay is performed in the assay container. Various embodiments of such assays are disclosed herein.

[0124] In many embodiments of the method, a multi-well plate is used for the assay. For example, aliquots of the test sample can be directly dispensed into the wells of the multi-well plate, a reproduction-deficient indicator phage can be added to the wells, and after a sufficient period for infection, lysis buffer and the substrate of the indicator portion (e.g., luciferase substrate of a luciferase indicator) can be added and the assay performed to detect the indicator signal. Some embodiments of the method can be performed on a filter plate. Some embodiments of the method can be performed with or without sample concentration before infection with the reproduction-deficient indicator phage.

[0125] The choice of plate (or any other container in which detection can be performed) can affect the detection procedure. For example, some plates may include colored or white backgrounds, which can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce a higher background signal. Plates of other colors may produce a lower background signal but also have slightly lower sensitivity. Additionally, one reason for background signal is light leakage from one aperture to another adjacent aperture. Some plates have white apertures, but the rest of the plate is black. This allows for a high signal within the aperture but prevents light leakage between apertures, thus reducing background. Therefore, the choice of plate or other measurement container can affect the sensitivity and background signal of the measurement.

[0126] The method according to embodiments of the invention may include various other steps to increase sensitivity. For example, as described in more detail herein, the method may include a step of washing the captured and infected microorganisms (such as bacteria) after the addition of the reproductively deficient indicator phage but before incubation to remove excess reproductively deficient indicator phage and / or luciferase or other reporter proteins contaminating the reproductively deficient indicator phage preparation.

[0127] The method according to embodiments of the present invention may include one or more sample preparation steps, which may be referred to as “sampling” or “sampling steps”. In some embodiments, the sample may be used directly in the method according to embodiments of the present invention without preparation, concentration, or dilution. For example, liquid samples may be directly measured. In other embodiments, the sample may be diluted or suspended in a solution, which may include, but is not limited to, buffer solutions or bacterial culture media. Solid or semi-solid samples may be suspended in a liquid by chopping, mixing, or immersing the solid in the liquid. In some embodiments, the sample should be maintained within a pH range that promotes the attachment of reproduction-deficient indicator phages to microorganisms of interest (such as bacteria of interest). In some embodiments, a preferred pH range may be a range suitable for the attachment of reproduction-deficient indicator phages to bacterial cells. The sample should also contain suitable concentrations of divalent and monovalent cations, including but not limited to Na+. +Mg 2+ and K + .

[0128] Throughout the assay, the sample is preferably maintained at a temperature that preserves the viability of any potentially present microorganisms of interest. During the step in which a replication-deficient indicator phage attaches to bacterial cells, the sample is preferably maintained at a temperature that promotes the activity of the replication-deficient indicator phage. Such a temperature is at least about 25°C and does not exceed 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 the binding or attachment of the replication-deficient indicator phage to the microorganism of interest.

[0129] Sampling can be performed in a variety of ways. 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 a culture medium in a test tube before sampling. In some embodiments, the solid support is dried before sampling. In some embodiments, the liquid sample is first incubated for a period of time (“incubation enrichment”), for example, less than 24 hours, less than 12 hours, less than 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In other embodiments, the sample can be enriched after the microorganism of interest has been captured on the solid support. In some embodiments, the solid support with microorganisms can be incubated in a growth medium to increase the number of microorganisms. This step is called “incubation enrichment”. In such implementations, depending on the sample type and size, the enrichment period can be 1, 2, 3, 4, 5, 6, 7 or up to 8 hours or longer.

[0130] In some implementations, the detection of microorganisms of interest can be performed without culturing samples as a means of increasing the microbial community. For example, in some implementations, the total time required for detection is less than 26.0, 25.0, 24.0, 23.0, 22.0, 21.0, 20.0, 19.0, 18.0, 17.0, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes, or less than 30 minutes. Minimizing the time to obtain results is crucial in various applications, such as the detection of pathogens in food and the environment.

[0131] The method according to embodiments of the invention may include steps aimed at infecting a reproductively deficient indicator phage with a microorganism of interest. For example, the reproductively deficient indicator phage may be contacted with the microorganism of interest by known methods, some of which are described herein. After contact with the microorganism of interest, the reproductively deficient indicator phage infects the microorganism of interest and expresses an indicator gene. The infection time, i.e., the time interval between the initial contact of the sample with the reproductively deficient indicator phage and the initiation of the detection step (e.g., adding the substrate of the enzymatic indicator moiety to the sample contacted with the reproductively deficient indicator phage), can vary depending on the type of reproductively deficient indicator phage and the concentration of the microorganism of interest in the sample. Using instruments in which the microorganism of interest (such as bacteria) is trapped on a solid support, the time required for infection can be significantly reduced; for example, the infection time can be 1 hour or less, whereas in standard assays (where no solid support is used to trap bacteria), infection typically takes at least 4 hours. In some embodiments, the infection time of 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. In some embodiments, the infection time is about 1 hour, about 2 hours, or about 3 hours.

[0132] A method according to an embodiment of the invention may include one or more steps associated with detecting a signal generated by an indicator. Indicators generated by expressing an indicator gene can be detected using known methods. For example, one or more signal-generating components may react 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 is achieved by reacting the enzyme with one or more substrates, or additionally with an enzyme and substrate, to generate a detectable reaction product. In another signal generation system, the indicator may be a fluorescent compound, where enzymatic operation of the indicator is not required to generate a detectable signal. Fluorescent molecules include, for example, fluorescein and rhodamine, and their derivatives and analogs, which are suitable for use as indicators in such systems. In yet another embodiment, the indicator portion may be a cofactor, in which case amplification of the detectable signal is achieved by reacting the cofactor with an enzyme and one or more substrates, or additionally with an enzyme and substrate, to generate a detectable reaction product. In some embodiments, the detectable signal is colorimetric. It should be noted that the selection of a specific indicator is not critical to this invention, but the indicator must be capable of generating a detectable signal on its own, or be detectable by the instrument, or be detectable in combination with one or more additional signal-generating components (such as an enzyme / substrate signal generation system). In some embodiments, the detection step will require the addition of a substrate containing an indicator enzyme to act. 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 last for 30 minutes or longer, and detection at various time points can be expected to optimize sensitivity. In some embodiments, a photometer reading can be initially acquired and readings can be acquired at intervals of 3 minutes, or 5 minutes, or 10 minutes, or 15 minutes until the reaction is complete.

[0133] Some embodiments of the method of the present invention include one or more steps related to a signal of a detection indicator, which may be referred to as "detection". Detection of the indicator gene product may include detecting its enzymatic activity. Detection of the indicator gene product may include detecting light emission or detecting optical density. In some embodiments, the chamber or container of the instrument in which the substrate is mixed with the test sample is transparent, such that any optical signals resulting from infection and subsequent incubation with the substrate are detectable without removing the sample from the chamber or container of the instrument. In this case, the signal can be detected through the walls of the chamber or container of the instrument. In some embodiments, the instrument or container containing the reacted sample is inserted into a device for detecting the generated signal. In other embodiments, the detection device is used to scan the instrument containing the reacted sample.

[0134] In some implementations, a photometer can be used to detect the reaction between an indicator (e.g., luciferase) and a substrate. Detection of RLU can be performed using a photometer, or other machines or devices can be used, some examples being derived from… (Madison, WI) 20 / 20 and In some implementations, a spectrophotometer, CCD camera, or CMOS camera can detect color changes and other light emissions. Absolute RLU is important for detection, but a high signal-to-background ratio (SBR) is also required (e.g., >2.0, >2.5, or >3.0) to reliably detect single cells or low cell numbers. The background signal can be obtained by measuring a control sample that does not contain microorganisms using the same procedure described above. In some implementations, detection of signals from reporter or indicator genes can include, for example, using devices employing photodiode or PMT (photomultiplier tube) technology. In some implementations, a handheld photometer can be used to detect the signal. Suitable handheld PMT photometers are available from 3M (Maplewood, MN). (Seattle, WA) and CHARM (Lawrence, MA). A suitable photodiode handheld photometer is available from (Camarillo, CA) and (Lansing, MI). Compared with conventional photometers (such as...) or Compared to 20 / 20, these handheld photometers typically produce much lower readings for the same sample, but multiple experiments have shown that the generated signals are sufficient for detection by these handheld photometers. The ability to use these handheld devices to detect microorganisms also provides convenience and flexibility that detection methods using conventional non-handheld detection devices often lack.

[0135] In some embodiments, the reproductively deficient indicator phage is genetically engineered to contain genes for an enzyme (such as luciferase) that is produced only after infection with a microorganism specifically recognized and infected by the phage. 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 with phage structural proteins that limit its copy number. Therefore, in some embodiments utilizing reproductively deficient indicator phages, the present invention includes a method for detecting a microorganism of interest, comprising the steps of: capturing at least one sample microorganism of interest; incubating said at least one microorganism of interest with a plurality of reproductively deficient indicator phages; allowing time for infection and expression of the soluble indicator moiety; and detecting said indicator moiety, wherein detection of said indicator moiety indicates the presence of said microorganism of interest in the sample.

[0136] For example, in some embodiments, the microorganisms of interest in the test sample can be captured by binding to the surface of a plate or by filtering the sample through a bacterial filter (e.g., a rotary or plate filter with a 0.45 μm pore size). In one embodiment, a reproductively deficient indicator phage is added directly to the captured sample onto the filter in a minimal volume. In one embodiment, the microorganisms captured on the filter or plate surface are subsequently washed once or multiple times to remove excess unbound reproductively deficient indicator phages. In one embodiment, a 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 tryptic soy broth, also referred to herein as TSB) may be added for further incubation time to allow sufficiently high levels of expression of genes encoding the indicator motif. In some implementations, the incubation step with the reproductively defective indicator phage only needs to be long enough to achieve a sufficient expression level of the gene encoding the indicator portion, said expression level being sufficient to allow a specified level of signal to be detected from the indicator portion (e.g., about 100-10000 RLU / s or about 200-5000 RLU / s), or sufficient to allow a specified level of signal-to-noise ratio (e.g., 1-500, 5-200, or 10-100).

[0137] In some implementations, aliquots of a test sample containing the microorganism of interest can be applied to a centrifuge column, and after infection with a reproductively defective indicator phage and optional washing to remove any excess reproductively defective indicator phage, the amount of soluble indicator detected will be proportional to the amount of reproductively defective indicator phage in the infected microorganism of interest.

[0138] The soluble indicator (e.g., luciferase) released into the surrounding liquid after bacterial lysis can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter, and the collected filtrate is measured in a new container (e.g., in a photometer) after the substrate of the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter. Thus, in an exemplary embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with a portion of the sample retained on the filter or bound to the surface of the plate. 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 the photometer. For example, in one embodiment, the present invention may include a method for detecting microorganisms of interest, comprising the steps of: infecting cells of the microorganism of interest trapped on a 96-well plate with a plurality of reproductively deficient indicator phages capable of expressing luciferase post-infection; washing away excess reproductively deficient indicator phages; adding LB broth and allowing time for the reproductively deficient indicator phages to express luciferase and lyse the microorganism of interest (e.g., 30-120 min, 60-120 min, or 80-100 min, e.g., about 90 min); and detecting indicator luciferase by adding a luciferase substrate and directly measuring luciferase activity in the 96-well plate, wherein the detection of luciferase activity indicates the presence of bacteria of interest in the sample.

[0139] In another embodiment, the invention may include a method for detecting a microorganism of interest, comprising the steps of: infecting cells in a liquid solution or suspension in a 96-well plate with a plurality of reproductively deficient indicator phages capable of expressing luciferase post-infection; allowing the reproductively deficient indicator phages time to express luciferase and lyse the microorganism of interest (e.g., 30-120 min, 60-120 min, or 80-100 min, e.g., about 90 min); and detecting the indicator luciferase by adding a luciferase substrate and directly measuring luciferase activity in the 96-well plate, wherein the detection of luciferase activity indicates the presence of the microorganism of interest in the sample. A capture step is not required in such embodiments. In some embodiments, the liquid solution or suspension may be a consumable test sample, such as a vegetable wash. In some embodiments, the liquid solution or suspension may be a vegetable wash fortified with concentrated LB broth, trypsin / trypsin peptone soy broth, peptone water, or nutrient broth. In some embodiments, the liquid solution or suspension may be bacteria diluted in LB broth.

[0140] In some embodiments, lysis of the microorganism of interest can occur before, during, or after the detection step. In some embodiments, infected, unly lysed cells can be detected after the addition of the luciferase substrate. Luciferase can leave the cell and / or the luciferase substrate can enter the cell without complete cell lysis. Therefore, for embodiments utilizing a rotary filter system, where the photometer analyzes only the luciferase released into the lysate (not the luciferase still inside the intact bacteria), lysis is required for detection. However, for embodiments utilizing a filter plate or 96-well plate in which the sample is in solution or suspension, where the photometer directly measures the original plate filled with both intact and lysed cells, lysis is not required for detection.

[0141] In some embodiments, the reaction between the indicator portion (e.g., luciferase) and the substrate can continue for 30 minutes or more, and detection at various time points can be used to optimize sensitivity. For example, in embodiments using a 96-well filter plate as a solid-phase support and luciferase as an indicator, a photometer reading can be initially acquired and then acquired at 10-minute or 15-minute intervals until the reaction is complete.

[0142] Surprisingly, high concentrations of reproductively deficient indicator phages used to infect test samples successfully achieved the detection of very low numbers of target microorganisms within a very short timeframe. In some embodiments, the incubation of the phage with the test sample requires only the time sufficient for a single phage lifetime. In some embodiments, the concentration of the reproductively deficient indicator phage used for this incubation step is greater than 7 x 10⁻⁶. 6 8x10 6 9x10 6 1.0x 10 7 1.1x10 7 1.2 x 10 7 1.3 x 10 7 1.4 x 10 7 1.5 x 10 7 1.6 x 10 7 1.7 x 10 7 1.8x10 7 1.9 x 10 7 2.0x 10 7 3.0x 10 7 4.0 x 10 7 5.0 x 10 7 6.0x 10 7 7.0x10 7 8.0x10 7 9.0x 10 7, or 1.0x 10 8 PFU / mL.

[0143] Embodiments of the method of the present invention can detect single microorganisms. Therefore, in some embodiments, the method can detect ≤10 microbial cells (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9 microorganisms) present in a sample. For example, in some embodiments, the reproduction-deficient indicator phage is highly specific to the bacteria of interest. In one embodiment, the reproduction-deficient indicator phage can distinguish the bacteria of interest in the presence of other types of bacteria. In some embodiments, the reproduction-deficient indicator phage can be used to detect a single type of bacteria in a sample. In some embodiments, the reproduction-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.

[0144] The large number of phages used for infection were previously thought to be associated with “external lysis,” which kills target cells and thus prevents the generation of useful signals. Purification of prepared breeding stock of reproductively deficient indicator phages, as described herein, can help mitigate this problem (e.g., purification by isodensity gradient ultracentrifugation with cesium chloride). This purification removes luciferase, any contaminating enzymes associated with reproductively deficient indicator phages, and also removes molted particles (particles that have lost DNA). Molted particles can lyse bacterial cells through “external lysis,” prematurely killing the cells and thus preventing the generation of indicator signals. Electron microscopy has demonstrated that crude phage lysates (i.e., before cesium chloride purification) can contain more than 50% molted phage. These molted particles can promote premature microbial death through the action of numerous phage particles piercing the cell membrane. Therefore, molted particles may contribute to the aforementioned problem, where high PFU concentrations have been reported to be detrimental. Furthermore, the purified formulation of the indicator phage with reproductive defects allows the assay to be performed without a washing step, enabling the assay to be performed without an initial concentration step. However, it should be understood that some embodiments of the method of the present invention include an initial concentration step, and in some embodiments, this concentration step allows for a shorter enrichment incubation time.

[0145] Some embodiments of the method of the present invention may further include confirmatory assays. Various assays are known in the art for confirming initial results, typically at a later point in time. For example, the sample may be cultured (e.g., as described in the examples). For assays, PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm preliminary results.

[0146] In some embodiments, in addition to utilizing infection source detection, the methods of the present invention combine the use of binders (e.g., antibodies) to purify and / or concentrate microorganisms of interest, such as bacteria of interest, from a sample. For example, in some embodiments, the present invention includes a method for detecting microorganisms of interest in a sample, comprising the steps of: capturing the microorganism (such as bacteria of interest) from the sample onto a prior support using a capture antibody specific to the microorganism of interest; incubating the sample with a reproductively deficient indicator phage infecting the bacteria of interest, wherein the reproductively deficient indicator phage contains an indicator gene inserted into a late gene region of the reproductively deficient indicator phage such that expression of the indicator gene after infection with the bacteria of interest results in the production of a soluble indicator protein product; and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the bacteria of interest in the sample.

[0147] For example, Figure 31 A hybrid immunophage (HIP) assay for detecting microorganisms of interest using a reproduction-deficient indicator phage according to an embodiment of the invention is described. First, a sample is applied to the wells 802 of a microtiter plate coated with a microorganism-specific antibody. The titration plate is then washed to promote binding of the microorganism of interest to the capture antibody 804. After sufficient time to allow for complete capture, a solution containing a microorganism-specific reproduction-deficient indicator phage is added to each sample 806. Incubation with the phage results in the binding and attachment of single or multiple phages to the captured microorganism 808. Finally, the sample is incubated to promote luciferase expression, leading to cell lysis and the release of soluble luciferase 810.

[0148] [System and Reagent Kit]

[0149] In some embodiments, the invention includes a system (e.g., an automated system or kit) comprising components for performing the methods disclosed herein. In some embodiments, a reproduction-deficient indicator phage is included in the system or kit according to the invention. The methods described herein can also utilize such a reproduction-deficient indicator phage system and / or kit. Given the small amounts of reagents and materials required to perform the methods, some embodiments described herein are particularly suitable for automation and / or kits. In some embodiments, each component of the kit may include a self-contained device that can be delivered from a first site to a second site.

[0150] In some embodiments, the present invention includes a system or kit for rapid detection of microorganisms of interest in a sample. In some embodiments, the system or kit may include components for incubating a sample with a reproductively deficient indicator phage specific to the microorganism of interest, wherein the reproductively deficient indicator phage includes an indicator portion, and components for detecting the indicator portion. In some embodiments of both the system and kit of the present invention, the reproductively deficient indicator phage is capable of specifically infecting bacteria of interest and includes an indicator gene inserted as an indicator portion into a late gene region of the reproductively deficient indicator phage, such that expression of the indicator gene during infection of the microorganism results in the production of a soluble indicator protein product. Some systems further include components for trapping the microorganism of interest on a solid support. In some embodiments, the system or kit may include: an instrument comprising a solid support (the solid support comprising a cell-binding component), and a signal detection component capable of detecting the indicator gene product produced by infecting a microorganism in a sample with a reproductively deficient indicator phage. In some embodiments, the signal detection component is a photometer, which may be a handheld device.

[0151] In other embodiments, the present invention includes a method, system, or kit for rapid detection of microorganisms of interest in a sample, comprising a reproductively deficient indicator phage component specific to said microorganism of interest, wherein said reproductively deficient indicator phage includes an indicator portion, and components for detecting said indicator portion. In some embodiments, the reproductively deficient indicator phage is highly specific to a particular microorganism (such as bacteria). In some embodiments, the reproductively deficient indicator phage can distinguish the microorganism of interest, such as bacteria, in the presence of other types of microorganisms. In some 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.

[0152] In some embodiments, the present invention may include a system or kit for rapid detection of microorganisms of interest in a sample, comprising an instrument having a first chamber containing a reproductively defective indicator phage. The instrument may further include a second chamber containing a substrate and / or a third chamber containing a culture medium. One or more of these chambers are sealed and separated from the rest of the instrument by a quick-acting seal, breaking which causes the contents of the chamber to exit the chamber and mix with the sample. Alternatively, the system or kit may further include separate containers containing the substrate and / or culture medium.

[0153] In some embodiments, the system and / or kit may further include a component for washing the captured microbial sample. Additionally or alternatively, the system and / or kit may further include a component for determining the amount of an indicator portion, wherein the amount of the indicator portion detected corresponds to the amount of microorganisms in the sample. For example, in some embodiments, the system or kit may include a photometer or other device for measuring luciferase activity.

[0154] In some implementations, the system and / or kit may include components for separating the microorganism of interest from other components in the 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 at least one step is performed by a liquid handling robot. In computerized systems, the system may be fully automated, semi-automated, or user-guided via computer (or some combination thereof).

[0155] Therefore, in some embodiments, the present invention may include a system or kit for rapid detection of microorganisms of interest in a sample, comprising: a component for incubating the sample with a reproductively deficient indicator phage specific to the microorganism of interest, wherein the reproductively deficient indicator phage includes an indicator portion; a component for trapping the microorganism of interest from the sample onto a solid support; a component for washing the trapped microorganism of interest to remove unbound reproductively deficient indicator phage; and a component for detecting the indicator portion. In some embodiments, the same component may be used for the trapping and / or incubation and / or washing steps (e.g., a filtration component). Some embodiments additionally include a component for determining the amount of the microorganism of interest in the sample, wherein the amount of the detected indicator portion corresponds to the amount of microorganism in the sample. Such a system may include various embodiments and sub-embodiments similar to those embodiments of the methods for rapid detection of microorganisms described above. In one embodiment, the microorganism is bacteria. In a computerized system, the system may be fully automated, semi-automated, or user-guided via computer (or some combination thereof). In some embodiments, the system may include a component for separating the microorganism of interest from other components in the sample.

[0156] In one embodiment, this disclosure includes a system or kit comprising 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 reproductively deficient indicator phages; components for lysing at least one infected microorganism to release reproductively deficient indicator phages present in the microorganism; and components for detecting the reproductively deficient indicator phage or, possibly with high sensitivity, a soluble protein encoded and expressed by the reproductively deficient indicator phage, wherein detection of the reproductively deficient indicator phage or a soluble protein product of the reproductively deficient indicator phage indicates the presence of the microorganism in the sample. The reproductively deficient indicator phage may be carrying... Indicator genes Indicator phages with reproductive defects.

[0157] In other embodiments, this disclosure may include a kit for rapid detection of a microorganism of interest in a sample, the system comprising: a component for incubating the sample with a reproductively deficient indicator phage specific to the microorganism of interest, wherein the reproductively deficient indicator phage includes an indicator portion; a component for trapping the microorganism of interest from the sample onto a solid support; a component for washing the trapped microorganism of interest to remove unbound reproductively deficient indicator phage; and a component for detecting the indicator portion. In some embodiments, the same component may be used for the trapping and / or incubation and / or washing steps. Some embodiments additionally include a component for determining the amount of the microorganism of interest in the sample, wherein the amount of the detected indicator portion corresponds to the amount of the microorganism of interest in the sample. Such a kit may include various embodiments and sub-implementations similar to those embodiments of the methods for rapid detection of microorganisms described above. In one embodiment, the microorganism is bacteria. In some embodiments, the kit may include a component for separating the microorganism of interest from other components in the sample.

[0158] The systems and kits disclosed herein include a variety of components. As used herein, the term "component" has a broad definition and includes any suitable instrument or assembly of instruments suitable for performing the methods. The components are not necessarily integrally connected or positioned relative to each other in any particular manner. This disclosure includes any suitable arrangement of the components relative to each other. For example, the components are not necessarily located in the same space. However, in some embodiments, the components are connected to each other within an integrated unit. In some embodiments, the same components can perform multiple functions.

[0159] Computer systems and computer-readable media

[0160] In some embodiments, this disclosure may include a system. The system may include at least some of the compositions of this disclosure. Furthermore, the system may include at least some components for performing the methods. In some embodiments, the system is configured as a kit. Therefore, in some embodiments, this disclosure may include a system for rapidly detecting microorganisms of interest in a sample. The system may include at least some of the compositions of this disclosure. Furthermore, the system may include at least some components for performing the methods. In some embodiments, the system is configured as a kit. Therefore, in some embodiments, this disclosure may include a system for rapidly detecting microorganisms of interest in a sample, including the instrument described above. For example, the instrument may include a first chamber containing a recombinant phage having a gene construct inserted into the phage genome, wherein the construct includes a promoter and an indicator gene; wherein the solid support includes a cell-binding component. In some embodiments, the system further includes a handheld detection device.

[0161] The system or any component thereof described in this technology may be embodied in the form of a computer system. Typical examples of a computer system include a general-purpose computer, a programmable microprocessor, a microcontroller, peripheral integrated circuit elements, and other means or configurations of means capable of implementing the steps of the methods constituting this technology.

[0162] A 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 memory. Memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include storage devices. Storage devices may be hard disk drives or removable storage drives (such as floppy disk drives, optical disk drives, etc.). Storage devices may also be other similar devices for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet via an I / O interface. The communication unit allows data to be transferred to and received from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that enables the computer system to connect to databases and networks (such as LANs, MANs, WANs, and the Internet). Therefore, the computer system can assist users by providing input through input devices and accessing the system through an I / O interface.

[0163] A computing device typically includes an operating system that provides executable program instructions for the comprehensive management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) storing the instructions, allowing the computing device to perform its intended functions when the server's processor executes the instructions. Suitable implementations of the operating system and the general functions of the computing device are known or commercially available and readily implemented by those skilled in the art, particularly in accordance with the disclosure herein.

[0164] A computer system executes a series of instructions stored in one or more storage elements to process input data. The storage elements may also hold data or other information as needed. The storage elements may be in the form of information sources present in the processor or physical storage elements.

[0165] The environment can include a wide variety of data storage and other storage media as described above. These can reside in various locations, such as storage media within one or more computer domains (and / or storage media residing in one or more computers) or remotely from any one or all computers in a network. In specific group implementations, information can reside in a storage area network (“SAN”) familiar to those skilled in the art. Similarly, any necessary files for performing functions that give a computer, server, or other network device can be stored locally and / or remotely, depending on the circumstances. Where the system includes computing devices, each such device can include hardware elements electrically connected via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., display device, printer, or microphone). Such systems can also include one or more storage devices, such as hard disk drives, optical storage devices, and solid-state storage devices such as random access memory (“RAM”) or read-only memory (“ROM”), as well as removable media devices, memory cards, flash cards, etc.

[0166] Such devices may also include computer-readable storage medium readers, communication devices (e.g., modems, network interface 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, which may be remote, local, fixed, and / or removable storage devices, and storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The systems and various devices also typically include numerous software applications, modules, services, or other elements residing within at least one working storage device, including operating systems and applications such as client applications or web browsers. It should be understood that alternative implementations can vary considerably from those described above. For example, custom hardware and / or specific elements may be used, implemented in hardware, software (including portable software such as applets), or both. Furthermore, connections to other computing devices, such as network input / output devices, may be employed.

[0167] Non-transient storage media and computer-readable media containing code or portions of code may include any suitable media known or used in the art, including storage media and communication media, such as, but not limited to, volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing and / or transmitting information (such as computer-readable instructions, data structures, program modules or other data), including RAM, ROM, EEPROM, flash memory or other storage technologies, CD-ROM, digital multi-disc (DVD) or other optical storage, magnetic cartridges, magnetic tape, disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by system devices. Based on this disclosure and the teachings provided herein, those skilled in the art will understand other ways and / or methods for implementing various embodiments.

[0168] Computer-readable media may include, but are not limited to, electronic, optical, magnetic, or other storage devices capable of providing a processor with computer-readable instructions. Other examples include, but are not limited to: floppy disks, CD-ROMs, DVDs, magnetic disks, memory modules, ROMs, RAMs, SRAMs, DRAMs, content-addressable memory ("CAM"), DDRs, 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), disk drives, and caches. The computing device may communicate with one or more external computer-readable media such as external hard drives or external DVD or Blu-ray drives.

[0169] As described above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronic devices such as PLCs, programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memory (PROMs), electronically programmable read-only memory (EPROMs or EEPROMs), or other similar devices.

[0170] The computing device includes a network interface. In some embodiments, the network interface is configured for communication via wired or wireless communication links. For example, the network interface may allow communication in a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface may allow communication in networks such as CDMA, GSM, UMTS, or other cellular communication networks. In some embodiments, the network interface may allow point-to-point connection with another device, such as via Universal Serial Bus (USB), FireWire 1394, serial or parallel, or similar interfaces. Some embodiments of a suitable computing device may include two or more network interfaces for communication in one or more networks. In some embodiments, the computing device may include data storage in addition to or in lieu of a network interface.

[0171] Some implementations of suitable computing devices may include or communicate with external or internal devices such as a mouse, CD-ROM, DVD, keyboard, monitor, speakers, one or more microphones, or any other input or output device. For example, the computing device may communicate with various user interface devices and displays. The display may use any suitable technology, including, but not limited to, LCD, LED, CRT, etc.

[0172] A set of instructions executed by a computer system may include various commands that instruct the processor to perform specific tasks (such as the steps constituting the methods of this technology). This set of instructions may be in the form of a software program. Furthermore, software may be a collection of independent programs, a program module containing a large program, or a portion of a program module, as in this technology. Software may also include modular programming in the form of object-oriented programming. The processor's processing of input data may be in response to user commands, the results of previous processing, or requests from another processor.

[0173] Although this disclosure has been made with reference to certain embodiments, numerous modifications, alterations, and changes are possible to the described embodiments without departing from the scope and spirit of this disclosure, which is defined by the appended claims. Therefore, it is intended that this disclosure be limited to the described embodiments, but rather have the full scope defined by the language of the following claims and their equivalents.

[0174]

Example

[0175] The following examples describe the detection of small numbers of cells or even single bacteria in a shortened time to obtain results, and are intended to be illustrative rather than limiting of this disclosure.

[0176] [Example 1. Establishment and isolation of a propagation-defective indicator phage from a phage specific to Escherichia coli O157:H7 serotype]

[0177] By using, for example Figure 3 The homologous recombination shown constructs a reproduction-deficient indicator phage specific to the E. coli O157:H7 serotype from a parental phage specific to the E. coli O157:H7 serotype. To generate the reproduction-deficient indicator phage, a method is used... The coding sequence replaces the coding sequence of the gp22 head scaffold protein of the parental bacteriophage. (Using...) Homologous recombinant plasmids of genes (flanked by bacterial genome sequences matching gp22) Figure 3 HR plasmids and plasmids containing gp22 expression cassettes Figure 3 pBAV.gp22 was transformed into E. coli O157:H7. Each plasmid underwent separate antibiotic selection to ensure the transformed bacteria contained both plasmids. These double-transformed bacteria were infected with a parental phage to allow homologous recombination with the HR plasmid, resulting in a deletion of the gp22 copy of the phage. The deleted portion was then provided by trans-p-coagulation of the plasmid encoding gp22. Following homologous recombination, a series of titration and enrichment steps were used to isolate the expression plasmid. Specific recombinant phages were developed. Large-scale production was conducted to obtain high-titer stock solutions of reproductively deficient indicator phages suitable for detection assays. Since the reproductively deficient indicator phage (designated CBA120Δgp22 NanoLuc) cannot reproduce in wild-type *E. coli* O157:H7, it was propagated in an engineered *E. coli* O157:H7 strain (“permissive” *E. coli* O157:H7 strain) transformed with a plasmid expressing the high-copy-value pUC-based gp22 scaffold protein. Figure 5 As shown. Figure 6 As shown in the growth curves, the indicator phage with reproductive defects successfully grew in permissive Escherichia coli O157:H7 strain. Phage particles were separated from contaminating luciferase proteins using cesium chloride isodense gradient centrifugation to reduce background.

[0178] [Example 2. Testing for indicator phages with specific reproductive defects against Escherichia coli O157:H7 serotype in a detection assay]

[0179] A strategy using the reproductively defective indicator phage CBA120Δgp22NanoLuc, specific to the Escherichia coli O157:H7 serotype, showed... Figure 7In Escherichia coli O157:H7, reproductively deficient indicator phages produce soluble luciferase. Due to the lack of the gp22 protein, reproductively deficient indicator phages cannot form phage heads. Reproductively deficient indicator phages do not produce viable progeny phages.

[0180] To evaluate the activity of the indicator phage CBA120Δgp22 NanoLuc, which is a reproductively defective phage, its activity was compared with that of CBA120 NanoLuc, a reproductively specific indicator phage of *Escherichia coli* O157:H7. The gene is inserted after gp23, and the major capsid protein gene is under the control of the T4 late-stage gene promoter. Logarithmic and stationary phase cultures of *E. coli* O157:H7 (ATCC 43888) were diluted to obtain the results when using 100 μl samples. Figure 8-11 The approximate number of CFUs is indicated on the x-axis. Each sample was infected with CBA120 NanoLuc or CBA120Δgp22 NanoLuc at 37°C for 2 hours. Lysis buffer and luciferase substrate were added, and the samples were read on a photometer. Five replicates were performed for each phage at each CFU level. The RLU value for each CFU was averaged. The average value at each CFU level was used to calculate the mean value and divided by the average value of the 0 CFU readings. Figure 8-11 The signal / background values ​​are plotted on the y-axis.

[0181] The above experiments were performed on *E. coli* O157:H7 cultures in the logarithmic growth phase, where bacterial cells typically produce high signal levels due to high transcription and protein expression levels. Results were... Figure 8 and 9 The results are shown in the image. The above experiments were performed on E. coli O157:H7 cultures still in the quiescent phase, where bacterial cells typically produce low signal levels due to low transcription and protein expression levels. Figure 10 and 11 As shown in [the image]. Figure 8 and 9 In the diagram, white bars represent results obtained with the indicator phage for reproductive defects (labeled CBA12.Δgp22.NL), and filled bars represent results obtained with the positive control (labeled CBA120NL). Figure 8-11 The indicator phages showing reproductive defects were comparable to the positive controls.

[0182] [Example 3. Testing the specificity of indicator phages for the reproductive defects of Escherichia coli O157:H7 serotype]

[0183] The specificity of a replication-deficient indicator phage specific to Escherichia coli O157:H7 serotype was tested. The assay was performed as described in previous examples to detect a range of bacteria. Results were... Figure 12 As shown in the figure, luciferase signals were generated during the detection of *E. coli* O157:H7 or engineered permissive *E. coli* O157:H7. No luciferase signals were detected during trial detection of non-target bacteria, including several *E. coli* serotypes.

[0184] [Example 4. Establishment and isolation of a propagation-defective indicator phage from the Salmonella-specific TSP1 phage]

[0185] By using, for example Figure 13 The homologous recombination shown constructs a Salmonella-specific, reproduction-deficient indicator phage from a Salmonella-specific parental phage. To generate the reproduction-deficient indicator phage, wild-type TSP1 was used... The coding sequence replaced the coding sequence of the gp22 head scaffold protein of the parental bacteriophage. Homologous recombination (HR) plasmids containing the NanoLuc gene (flanked by bacterial genome sequences matching those flanking gp22) were used (i.e., the gp21 head core and protease, and the gp23 major capsid protein). Figure 13 Salmonella ATCC 19585 was transformed. The transformed bacteria were infected with parental phages to allow homologous recombination with the HR plasmid, resulting in the deletion of the gp22 copy of the phage. This simultaneously created a reproductively defective mutant and inserted an indicator gene (i.e., This process was used to form indicator phages with reproductive defects. Infected cells produced a mixture of wild-type and recombinant phages at a ratio of approximately 1:8. Co-infection with wild-type phages supported recombinant replication by trans-supplementing the missing gp22 gene. Following homologous recombination, a series of titration and enrichment steps were used to isolate the expressed gene. Specific recombinant phages were developed. Large-scale production was conducted to obtain high-titer stock solutions of reproductively deficient indicator phages suitable for detection assays. Since the reproductively deficient indicator phage (designated TSP1.Δgp22 NanoLuc) could not reproduce in wild-type Salmonella 19585, it was propagated in engineered Salmonella strains transformed with plasmids expressing the gp22 head scaffold protein using high-copy pUC-based plasmids (“permissive” Salmonella strains), such as... Figure 14 As shown in the image.

[0186] The isolated TSP1.Δgp22.NanoLuc plaques were suspended in TMS buffer and inoculated into wild-type or permissive Salmonella 19585 cultures, and incubated at 37°C for 3 hours. NanoGlo (NanoGlo) assays were performed on 10 μl samples. ) Determination. TSP1.Δgp22.NanoLuc infection of wild-type and permissive Salmonella both produced high signals above the background (100 RLU / s). Figure 15 ).

[0187] [Example 5. Testing the detection limit of TSP1 indicator phage, which is specifically degeneratively defective to Salmonella]

[0188] To assess the detection limit of the indicator phage TSP1.Δgp22.NanoLuc reproductive defect in stationary Salmonella, Salmonella Typhimurium ATCC 19585 was grown for 18–20 hours until the stationary phase. The stationary culture was diluted in TSB and... Figure 16 The plate layout shown in the image was used to transfer cells to a 96-well plate. TSP1.Δgp22.NanoLuc phage was added to a stationary Salmonella culture and incubated at 37°C for 2 hours. After phage infection, lysis buffer, assay buffer, and substrate were added, and the plate was read for 1 second on a photometer. Results are shown in... Figure 16 middle.

[0189] To assess the detection limit of the indicator phage TSP1.Δgp22.NanoLuc reproductive defect in logarithmic-phase Salmonella, Salmonella Typhimurium ATCC 19585 was grown for 18–20 hours to the stationary phase. The stationary cell culture was then diluted in TSB and grown to the early logarithmic phase. The logarithmic-phase Salmonella culture was then diluted in TSB and grown according to… Figure 17 The layout shown in the image shows the transfer of cells to a 96-well plate. TSP1.Δgp22.NanoLuc phage was added to the Salmonella logarithmic phase culture and incubated at 37°C for 2 hours. After phage infection, lysis buffer, assay buffer, and substrate were added, and the plate was read for 1 second on a photometer. Results are shown in... Figure 17 middle.

[0190] [Example 6. Establishment and isolation of a propagation-defective SEA1 indicator phage from a Salmonella-specific phage]

[0191] By using, for example Figure 18 The homologous recombination shown constructs a Salmonella-specific, reproduction-deficient indicator phage from a Salmonella-specific parental phage. To generate the reproduction-deficient indicator phage, wild-type SEA1 was used... The coding sequence replaced the coding sequence of the gp84 substrate wedge subunit protein of the parental bacteriophage. Homologous recombination (HR) plasmids containing the NanoLuc gene (flanked by bacterial genome sequences matching those flanking gp84) were used (i.e., gp83 head completion protein and gp85 substrate pore subunit and tail lysozyme). Figure 18 Salmonella 27869 was transformed. These transformed bacteria were then infected with a parental phage to allow homologous recombination with the HR plasmid, resulting in a deletion of the gp84 copy of the phage. This simultaneously created a reproductively defective mutant and inserted an indicator gene (i.e., This process was used to create indicator phages with reproductive defects. Infected cells produced a mixture of wild-type and recombinant phages. Co-infection with wild-type phages supported recombinant replication by trans-supplementing the missing gp84 gene. Following homologous recombination, a series of titration and enrichment steps were used to isolate the expression. Specific recombinant phages were developed. Large-scale production was conducted to obtain high-titer stock solutions of reproductively deficient indicator phages suitable for detection assays. Since the reproductively deficient indicator phage (designated SEA1.Δgp84.NanoLuc) could not reproduce in wild-type Salmonella 27869, it was propagated in engineered Salmonella strains transformed with plasmids expressing the gp84 slice wedge subunit protein at high copy numbers (“permissive” Salmonella strains), such as... Figure 19 As shown in the image.

[0192] [Example 7. Testing SEA1 indicator phages with specific reproductive defects against wild-type and permissive Salmonella]

[0193] Compare time-dependent infection of wild-type Salmonella 27869 with 27869 allowable cells transformed with pUC57.trans.SEA1.gp84. (1.0 x 10⁻⁶) 6 Wild-type Salmonella 27869 cells / well (in 200 μl TSB) or 27869 permissible cells transformed with pUC57.trans.SEA1.gp84 (in 200 μl TSB+carb) were incubated with recombinant phage (MOI 0.1) in triplicate. NanoGlo assays were performed on 10 μl samples at 37°C for 4 hours. In wild-type Salmonella, the signal produced by the reproductively deficient recombinant phage reached plateau early and was weak, indicating that the phage lacked sustained growth in wild-type Salmonella. Figure 20 However, the signal in permissible Salmonella continued to increase over time, indicating multiple rounds of infection and continued growth. Figure 20 ).

[0194] The next step is to conduct time-series infection with wild-type Salmonella strains 7001, 8326, 13076, and 27869. 1.0 x 10 6 Each wild-type Salmonella strain per cell / well was incubated with a recombinant phage (MOI 0.1) in 100 μl TSB. NanoGlo assays were performed on 10 μl samples at 37°C for 0, 1, 2, and 5 hours. In wild-type Salmonella, the signal produced by the replication-deficient recombinant phage reached a plateau early and was weak. Figure 21 ).

[0195] The replication of SEA1.Δgp84.NanoLuc phage on wild-type Salmonella strains was assessed by plaque assay of 5-hour wild-type cultures (40 μl culture). No plaques were formed in cultures of wild-type Salmonella strains 7001, 8326, 13076, and 27869. Figure 22 This confirmed the absence of SEA1.Δgp84.NanoLuc replication in wild-type Salmonella strains.

[0196] [Example 8. Testing the detection limit of SEA1 indicator phage, which is specifically degeneratively defective to Salmonella]

[0197] To assess the detection limit of the indicator phage SEA1.Δgp84.NanoLuc for reproductive defects, *Salmonella Newport* ATCC 27869 was transformed with AmpR puc57.SEA1.Trans gp84. Logarithmic phase cultures were diluted in TSB and... Figure 23 The plate layout shown in the image shows the transfer of cells to a 96-well plate. SEA1.Δgp84.NanoLuc phage was added to the Salmonella logarithmic phase culture and incubated at 37°C for 2 hours. After phage infection, lysis buffer, assay buffer, and substrate were added, and the plate was read for 1 second on a photometer. Results are shown in... Figure 23 middle.

[0198] To assess the detection limit of the indicator phage SEA1.Δgp84.NanoLuc proliferative defect in stationary Salmonella, *Salmonella choleraesuis* ATCC 27869 was grown for 18–20 hours to the stationary phase. The stationary cells were then diluted in TSB and analyzed according to… Figure 24 The plate layout shown in the image shows the transfer of cells to a 96-well plate. SEA1.Δgp84.NanoLuc phage was added to a stationary Salmonella culture and incubated at 37°C for 2 hours. After phage infection, lysis buffer, assay buffer, and substrate were added, and the plate was read for 1 second on a photometer. Results are shown in... Figure 24 middle.

[0199] To assess the detection limit of the indicator phage SEA1.Δgp84.NanoLuc proliferative defect in logarithmic-phase Salmonella, *Salmonella choleraesuis* ATCC 27869 was grown for 18–20 hours to the stationary phase. The stationary cells were then diluted in TSB and grown to the early logarithmic phase. The logarithmic-phase culture was then diluted in TSB and grown according to… Figure 25 The plate layout shown in the image shows the transfer of cells to a 96-well plate. SEA1.Δgp84.NanoLuc phage was added to a stationary Salmonella culture and incubated at 37°C for 2 hours. After phage infection, lysis buffer, assay buffer, and substrate were added, and the plate was read for 1 second on a photometer. Results are shown in... Figure 25 middle.

[0200] [Example 9. Testing of SEA1 indicator phage with specific reproductive defects against Salmonella spp. in a detection assay]

[0201] To evaluate the activity of the indicator phage SEA1.Δgp84.NanoLuc, which exhibits reproductive defects, its activity was compared with that of SEA1 NanoLuc, a reproductive indicator phage specific to Salmonella. The gene was inserted after gp84. Each sample was infected with SEA1 NanoLuc or SEA1.Δgp84.NanoLuc at 37°C for 2 and 4 hours. Lysis buffer and luciferase substrate were added, and the samples were read on a spectrophotometer. Five replicates were performed for each phage at each CFU level, as shown below. Figure 26A As shown in the image. The RLU value for each CFU is displayed. Figure 26B (2-hour infection) and Figure 26C (Infection within 4 hours)

[0202] This instruction manual also includes the following:

[0203] 1. A recombinant phage comprising an indicator gene in a late gene region of the phage's genome, wherein the recombinant phage is reproductively defective and wherein the recombinant phage is capable of specifically infecting microorganisms of interest.

[0204] 2. The recombinant phage according to Embodiment 1, wherein the phage is reproductively defective due to alterations or deletions of late genes required for viral particle assembly.

[0205] 3. The recombinant phage according to embodiment 1, wherein the indicator gene is inserted into the sequence of the late gene of the recombinant phage, such that the late gene is nonfunctional and the recombinant phage becomes reproductively defective.

[0206] 4. The recombinant phage according to embodiment 1, wherein the indicator gene replaces at least a portion of the sequence of the late gene of the recombinant phage, making the recombinant phage reproductively defective, wherein the late gene is required for viral particle assembly.

[0207] 5. The recombinant phage according to Embodiment 1, wherein the recombinant phage is derived from a phage specific to Escherichia coli, Salmonella, Listeria, or Staphylococcus.

[0208] 6. The recombinant phage according to Embodiment 1, wherein the late gene is required for viral particle assembly.

[0209] 7. A composition comprising at least two recombinant phages, each of the two recombinant phages containing an indicator gene in a late gene region of the genome of the phage, wherein the recombinant phage is reproductively defective, and wherein the recombinant phage is capable of specifically infecting one or more microorganisms of interest.

[0210] 8. The composition according to embodiment 7, wherein each of the at least two recombinant phages contains a different indicator gene.

[0211] 9. The composition according to embodiment 8, wherein each of the at least two recombinant phages is capable of specifically infecting different microorganisms of interest.

[0212] 10. The composition according to embodiment 8, wherein the at least two recombinant phages are capable of infecting a variety of microorganisms of interest.

[0213] 11. The composition according to embodiment 7, wherein the microorganism of interest includes at least one of Escherichia coli, Salmonella, Listeria, and Staphylococcus.

[0214] 12. The composition according to embodiment 10, wherein the plurality of microorganisms of interest comprises at least two different classes of bacteria.

[0215] 13. The composition according to embodiment 12, wherein the at least two different classes of bacteria include 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.

[0216] 14. A method for preparing recombinant bacteriophages, comprising:

[0217] Select parental bacteriophages that specifically infect the target microorganism;

[0218] The genes of the parent phage were altered to produce a recombinant, reproductively defective phage;

[0219] Transform an engineered strain of a target microorganism with a homologous recombination (HR) plasmid, the engineered strain being able to express the product of a mutated gene in the reproductively defective phage, the homologous recombination (HR) plasmid comprising an indicator gene and an HR sequence flanking the indicator gene and homologous to a desired sequence in the parental phage;

[0220] Infect the transformed target microorganism with the parental phage or the reproductively defective parental phage, enabling HR to occur between the HR plasmid and the genome of the parental phage or the recombinant reproductively defective phage; and

[0221] Isolate specific clones of recombinant phages, wherein the specific clones are reproductively defective products capable of expressing the indicator gene.

[0222] 15. The method according to embodiment 14, wherein the alteration of the parental phage gene to produce the reproductively defective phage is achieved by an HR occurring between the HR plasmid and the genome of the parental phage, wherein the parental phage gene is altered by replacing at least a portion of the parental phage with the indicator gene.

[0223] 16. The method according to embodiment 14 further includes generating an engineered strain of the target microorganism.

[0224] 17. The method according to embodiment 16, wherein the engineered strain for generating the target microorganism comprises transforming the target microorganism using a plasmid encoding a gene that is altered in the recombinant reproductively defective phage.

[0225] 18. The method according to embodiment 14, wherein the transformation of the engineered strain further comprises transforming the engineered strain using a trans plasmid.

[0226] 19. The method according to embodiment 14 further includes, prior to the transformation, preparing the homologous recombinant plasmid containing the indicator gene.

[0227] 20. The method according to embodiment 14, wherein the isolation of a specific clone of the recombinant phage comprises performing a limiting dilution assay to isolate a clone demonstrating expression of the indicator gene, the specific clone being a reproductively defective product capable of expressing the indicator gene.

[0228] 21. The method according to embodiment 14, wherein the recombinant phage is derived from a phage specific to Escherichia coli, Salmonella, Listeria, or Staphylococcus.

[0229] 22. A method for detecting microorganisms of interest in a sample, comprising:

[0230] The sample was incubated with the recombinant phage described in Embodiment 1; and

[0231] The product of the indicator gene is detected, wherein a positive detection of the product of the indicator gene indicates that the microorganism of interest is present in the sample.

[0232] 23. The method according to embodiment 22, wherein the sample is a food sample, an environmental sample, a water sample, or a commodity sample.

[0233] 24. The method according to embodiment 22, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single microorganism in the sample.

[0234] 25. The method according to embodiment 22, wherein the microorganism of interest is Escherichia coli, or Salmonella, or Listeria, or Staphylococcus.

[0235] 26. The method according to embodiment 22, wherein the microorganism of interest is Salmonella.

[0236] 27. A kit for detecting a microorganism of interest in a sample, comprising the recombinant phage of embodiment 1 and a substrate for reacting with the product of the indicator gene to detect the product of the indicator gene.

[0237] 28. A system for detecting a microorganism of interest, comprising the recombinant phage described in Embodiment 1 and components for detecting the product of the indicator gene.

Claims

1. A recombinant bacteriophage comprising an indicator gene in a late gene region of the genome of the bacteriophage, wherein the recombinant bacteriophage is propagation deficient, and wherein the recombinant bacteriophage is capable of specifically infecting a microorganism of interest.

2. The recombinant bacteriophage of claim 1, wherein the bacteriophage is propagation deficient due to an alteration or deletion of a late gene required for virion assembly.

3. The recombinant bacteriophage of claim 1, wherein the indicator gene is inserted into the sequence of a late gene of the recombinant bacteriophage such that the late gene is nonfunctional and the recombinant bacteriophage is propagation deficient.

4. The recombinant bacteriophage of claim 1, wherein the indicator gene replaces at least a portion of the sequence of a late gene of the recombinant bacteriophage such that the recombinant bacteriophage is propagation deficient, wherein the late gene is required for virion assembly.

5. The recombinant bacteriophage of claim 1, wherein the recombinant bacteriophage is derived from a bacteriophage specific for E. coli, or Salmonella, or Listeria or Staphylococcus.

6. The recombinant bacteriophage of claim 1, wherein the late gene is required for virion assembly.

7. A composition comprising at least two recombinant bacteriophages each comprising an indicator gene in a late gene region of the genome of the bacteriophage, wherein the recombinant bacteriophage is propagation deficient, and wherein the recombinant bacteriophage is capable of specifically infecting one or more microorganisms of interest.

8. The composition of claim 7, wherein each of the at least two recombinant bacteriophages comprises a different indicator gene.

9. The composition of claim 8, wherein each of the at least two recombinant bacteriophages is capable of specifically infecting a different microorganism of interest.

10. The composition of claim 8, wherein the at least two recombinant bacteriophages are capable of infecting a plurality of microorganisms of interest.

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