CN108884498A - Method and system for rapid detection of microorganisms using infectious agents - Google Patents

Abstract

Disclosed herein is the method and systems for microorganism in quick test sample.The bacteriophage of genetic modification is also disclosed, it includes the indicators in late gene area.The specificity of bacteriophage (such as CBA120) allows to detect specified microorganisms, such as Escherichia coli O 157:H7, and indication signal can be expanded to optimize measurement sensitivity.

Description

Method and system for rapid detection of microorganisms using infectious agents

related application

This application claims priority to U.S. Provisional Patent Application No. 62/280,043, filed January 18, 2016, and U.S. Provisional Patent Application No. 62/280,465, filed January 19, 2016. This application also claims priority to US Application No. 15/263,619, filed September 13, 2016.

References to sequence listings submitted as text files via EFS-WEB

The official copy of the sequence listing is submitted electronically via EFS-Web as the sequence listing in ASCII format, with the file name 1035526_ST25.txt, created on January 17, 2017, 7 kilobytes in size, and submitted at the same time as the description. The Sequence Listing contained in this document in ASCII format is part of the specification and is hereby incorporated by reference in its entirety.

field of invention

The present invention relates to methods and systems for detecting microorganisms using infectious agents.

background

There is a strong interest in improving the speed and sensitivity of detection of bacteria, viruses and other microorganisms in biological, food, water and clinical samples. Microbial pathogens can cause considerable morbidity in humans and livestock, as well as enormous economic losses. In addition, in view of the pathogenic or devastating diseases caused by ingestion of food contaminated by certain microorganisms (for example, E. coli (E. coli) or Salmonella (Salmonella spp.) And the Centers for Disease Control and Prevention (CDC) is a high priority.

Traditional microbiological testing for bacterial detection relies on nonselective and selective enrichment cultures followed by plating on selective media and further testing to confirm suspected colonies. Such a procedure may take several days. Various fast methods have been studied and introduced practices to shorten the time requirement. However, these methods have drawbacks. For example, techniques involving direct immunoassays or gene probes often require an overnight enrichment step to achieve adequate sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step, and are therefore capable of very high sensitivity and selectivity; however, the size of samples that can economically be tested by PCR is limited. With dilute bacterial suspensions, most of the small sub-samples will be cell-free and thus still require purification and/or lengthy enrichment steps.

The time required for traditional bioconcentration is governed by the growth rate of the sample's target bacterial population, the influence of the sample matrix, and the desired sensitivity. In practice, most high-sensitivity methods use overnight incubation and take approximately 24 hours in total. Due to the time required for incubation, these methods can take up to three days depending on the organism to be assayed and the source of the sample. This lag time is often inappropriate because contaminated food, water (or other products) may have entered livestock or people. Furthermore, the increase in antibiotic-resistant bacteria and biodefense considerations have made the rapid identification of bacterial pathogens in water, food, and clinical samples a very high priority worldwide.

Therefore, there is a need for rapid, simple and sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms.

overview

Embodiments of the invention include compositions, methods, systems and kits for microbial detection. The present invention can be embodied in various ways.

In some aspects, the invention includes recombinant phage comprising an indicator gene inserted into the late gene region of the phage genome. In some embodiments, the recombinant phage is a genetically modified CBA120 genome. In some embodiments, the recombinant phage is a genetically modified T4-like or ViI-like phage genome. In some embodiments, the recombinant phage specifically infects E. coli O157:H7. In one embodiment, the recombinant phage can differentiate E. coli O157:H7 in the presence of more than 100 other types of bacteria.

In some embodiments of the recombinant indicator phage, the indicator gene may be codon-optimized and may encode a soluble protein product that produces an intrinsic signal or a soluble enzyme that produces a signal upon reaction with a substrate. Some recombinant phage also contain a codon-optimized untranslated region upstream of the indicator gene, where the untranslated region includes the phage late gene promoter and ribosome entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene may be a naturally occurring gene such as Oplophorus luciferase, Firefly luciferase, Lucia luciferase or Renilla luciferase, or it may be a genetically engineered gene.

Also disclosed herein are methods for preparing recombinant indicator phages. Some embodiments include selecting wild-type phages that specifically infect target pathogenic bacteria; preparing homologous recombination plasmids/vectors containing indicator genes; transforming homologous recombination plasmids/vectors into target pathogenic bacteria; infecting transformed target pathogenic bacteria with selected wild-type phages , thereby allowing homologous recombination between the plasmid/vector and the phage genome; and isolating specific clones of the recombinant phage. In some embodiments, the wild-type phage selected is CBA120. In some embodiments, the selected wild-type phage is T4-like or ViI-like.

In some embodiments, making a homologous recombination plasmid/vector comprises determining the native nucleotide sequence in the late region of the genome of the selected phage; annotating the genome and identifying the major capsid protein gene of the selected phage; designing the major capsid protein A homologous recombination sequence downstream of the gene, where the sequence contains a codon-optimized indicator gene; and the sequence designed for homologous recombination is incorporated into the plasmid/vector. The step of designing the sequence may include inserting an untranslated region upstream of the codon-optimized indicator gene, which includes the phage late gene promoter and ribosome entry site. Thus, in some methods, the homologous recombination plasmid contains a codon-optimized untranslated region upstream of the indicator gene, which includes the phage late gene promoter and ribosome entry site.

Some embodiments of the invention are compositions comprising a recombinant indicator phage as described herein. For example, a composition can include one or more wild-type or genetically modified infectious agents (eg, bacteriophage) and one or more indicator genes. In some embodiments, the composition may include a mixture of different indicator phages, which may encode and express the same or different indicator proteins.

In some embodiments, the present invention includes a method for detecting a target microorganism in a sample comprising the step of incubating the sample with a recombinant phage that infects the target microorganism, wherein the recombinant phage contains an indicator gene inserted into the late gene region of the phage such that in The expression of the indicator gene during the phage replication process after host bacterial infection produces a soluble indicator protein product, and the method further includes detecting the indicator protein product, wherein the positive detection of the indicator protein product indicates that the target microorganism exists in the sample.

In some embodiments of the method of making a recombinant indicator phage, the wild-type phage is CBA120 and the target pathogen is E. coli O157:H7. In some embodiments, isolating specific clones of recombinant phage comprises a limiting dilution assay for isolating clones demonstrating expression of the indicated gene.

Other aspects of the invention include methods for detecting bacteria (e.g. E. coli O157:H7) in a sample comprising the steps of incubating the sample with a recombinant phage derived from CBA120 and detecting an indicator protein product produced by the recombinant phage, wherein Positive detection of the indicator protein product indicates the presence of E. coli O157:H7 in the sample. Samples can be food, environmental, water, commercial or clinical samples. In some embodiments, the sample includes beef or vegetables.

In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions conducive to enriched growth for a period of 9 hours or less, 8 hours or less, 7 hours or less Short, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In some embodiments, the total time to result is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours. In some embodiments, the ratio of signal to background produced by detection of the indicator is at least 2.0 or at least 2.5. In some embodiments, the method detects as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 , 60, 70, 80, 90 or 100 of specific bacteria.

Additional embodiments include systems and kits for the detection of E. coli O157:H7, wherein the systems or kits include recombinant phage derived from CBA120. Some embodiments also include a substrate for reacting with the indicator to detect the soluble protein product expressed by the recombinant phage. These systems or kits may include the features described for the phage, compositions and methods of the invention. In other embodiments, the present invention includes non-transitory computer readable media for use with methods or systems according to the present invention.

Description of drawings

The invention can be better understood by reference to the following non-limiting drawings.

Figure 1 shows a portion of the genome of wild-type CBA120 phage and in particular the annotated late gene region.

Figure 2 shows an embodiment of a plasmid designed for homologous recombination with the CBA120 phage genome. The capsid protein gp23(ORF187) is considered to represent the major capsid protein. Since this virion protein is expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels as long as the late gene promoter and/or other similar control elements are used.

FIG. 3 shows an embodiment of homologous recombination of the wild-type CBA120 genome in FIG. 1 with the plasmid shown in FIG. 2 .

Figure 4 depicts the isolation of recombinant phage from a mixture of wild-type and recombinant phage derived from transformation of target bacteria with a plasmid carrying a sequence designed for recombination in a homologous manner with the native phage genome, followed by wild-type The phage infect transformed bacteria to allow homologous recombination. A series of sequential infection and dilution steps allow the identification and isolation of recombinant phage expressing the indicator/reporter gene.

Figure 5 is an electron micrograph of one embodiment of the recombinant indicator phage CBA120NanoLuc phage.

Figure 6 depicts detection of bacterial cells using indicator phage encoding a soluble reporter gene (eg, luciferase) by detecting luciferase produced by replication of the indicator phage during bacterial cell infection, according to one embodiment of the present invention.

Figure 7 shows the infection of samples with known numbers of cells using different phage concentrations of ^{CBA120NanoLuc} to detect pathogenic bacteria, with 106 phage/mL yielding the highest signal-to-background ratio.

Figure ⁸ shows that repetitions of experiments using 106 phage/mL of CBA120NanoLuc to infect samples with known numbers of cells showed a significant difference between the signal from a single cell and the signal from 0 cells, 2 cells or more difference.

Figure 9 shows that the signal-to-background ratio for the experiment shown in Figure 8 was greater than 2.0.

Figure 10 shows the relative light units (RLU) and signal-to-background ratio for the detection of E. coli O157:H7 in 1 mL concentration samples from 25 g ground beef when assayed after 5, 6 and 7 hours of enrichment.

Figure 11 summarizes the detection of E. coli O157:H7 in 1 mL concentration samples from 25 g of ground beef as shown in Figure 10 and confirms the results using the second method.

Figure 12 shows the RLU and signal-to-background ratio for the detection of E. coli O157:H7 in a 10 mL concentrated sample from 25 g of ground beef when assayed 5 hours after enrichment and confirms the results using a second method.

Figure 13 shows the RLU and signal to background ratio for the detection of E. coli O157:H7 in 1 mL concentration samples from 125 g of ground beef when assayed after 7, 8 and 9 hours of enrichment.

Figure 14 shows the RLU and signal-to-background ratio for the detection of E. coli O157:H7 in 10 mL concentration samples from 125 g of ground beef when assayed after 7, 8 and 9 hours of enrichment.

Figure 15 summarizes the detection of E. coli O157:H7 in 1 mL concentration samples from 125 g of ground beef as shown in Figure 13 and confirms the results using the second method.

Figure 16 summarizes the detection of E. coli O157:H7 in a 10 mL concentration sample from 125 g of ground beef as shown in Figure 14 and confirms the results using the second method.

Figure 17 shows the RLU and signal-to-background ratio for detection of E. coli O157:H7 in 100 mL of spinach wash that was filtered and subjected to the filtration assay format and confirmed the results using a secondary method.

Detailed description of the invention

Disclosed herein are compositions, methods, and systems that demonstrate surprising sensitivity for detecting target microorganisms in assay samples (eg, biological, blood, water, and clinical samples). In assays performed without the use of enrichment cultures or, in some embodiments, with minimal incubation times during which the microorganisms may potentially multiply, genetically modified infectious agents may be used for periods of time shorter than previously thought possible detection in the implementation. Also surprising was the success of using potentially high multiplicity of infection (MOI) or high concentrations of plaque forming units (PFU) for incubation with test samples. Such high phage concentrations (PFU/mL) were previously said to be detrimental to bacterial detection assays, as they were said to cause "lysis from outside". However, high concentrations of phage can facilitate discovery, binding, and infection of low numbers of target cells.

Compositions, methods, systems and kits of the invention may include infectious agents for the detection of such microorganisms. In certain embodiments, the invention includes compositions comprising a recombinant phage having an indicator gene inserted into the late gene region of the phage. In certain embodiments, expression of the indicator gene during phage replication following infection of the host bacterium results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene (ie, class III) region of the phage. The phage can be derived from T7, T4, T4-like, ViI, ViI-like (or ViI virus according to GenBank/NCBI), CBA120 or another wild type or engineered phage.

In some aspects, the invention includes methods of detecting a target microorganism. The method can use infectious agents for detection of target microorganisms. For example, in certain embodiments, the target microorganism is a bacterium and the infectious agent is a bacteriophage. Thus, in certain embodiments, the method may comprise detecting a bacterium of interest in a sample by incubating the sample with a recombinant phage that infects the bacterium of interest. In certain embodiments, the recombinant phage includes an indicator gene. In certain embodiments, the indicator gene can be inserted into the late gene region of the phage such that expression of the indicator gene during phage replication following infection of the host bacterium results in production of the indicator protein product. The method can include detecting an indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the indicator protein is soluble.

In some embodiments, the invention may include a system. The system may contain at least some compositions of the invention. Additionally, the system may include at least some of the components for performing the method. In certain embodiments, the system is formulated as a kit. In certain embodiments, the present invention may include a system for the rapid detection of a target microorganism in a sample comprising: components for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent includes an indicator moiety ; and components for detecting the indicating moiety. In still other embodiments, the invention includes software for use with the methods or systems described.

Accordingly, some methods of the present invention address the need to amplify the detectable signal indicative of the presence of bacteria by using phage-based methods. In certain embodiments, as few as a single bacterium are detected. The principles applied herein can be applied to the detection of various microorganisms. Due to the myriad binding sites for infectious agents on the surface of microorganisms, the ability to generate a hundred or more infectious atom generations during infection, and the potential for high-level expression of the encoded indicator moiety, the infectious agent or indicator moiety can be more effective than the microorganism itself. detected more quickly. In this way, embodiments of the invention can achieve enormous signal amplification from even a single infected bacterium.

Aspects of the invention take advantage of the high specificity of binding agents that can bind specific microorganisms, such as binding components of infectious agents, as a means of detecting and/or quantifying specific microorganisms in a sample. In some embodiments, the invention takes advantage of the high specificity of infectious agents such as phages.

In some embodiments, detection is accomplished by an indicator moiety associated with a binding agent specific for the target microorganism. For example, an infectious agent may include an indicator moiety, such as a gene encoding a soluble indicator. In some embodiments, the indicator agent can be encoded by an infectious agent, such as a phage, and the phage is designated as an indicator phage.

Some embodiments of the invention disclosed and described herein take advantage of the discovery that individual microorganisms are capable of binding specific recognition agents, such as bacteriophages. After phage infection and replication, progeny phage can be detected by the indicator moieties expressed during phage replication. This principle, based on specific recognition of microbial surface receptors, allows amplification of indicator signals from one or a few cells. For example, by exposing even a single bacterial cell to multiple phages, thereafter allowing amplification of the phages during replication and high level expression of the encoded indicator gene product, the indicator signal is amplified so that individual bacteria can be detected.

Embodiments of the methods and systems of the present invention can be applied to the detection and quantification of various microorganisms (e.g., bacteria, fungi, yeasts) in a variety of settings, including but not limited to the detection of pathogens from food, water, clinical and commercial samples . The method of the present invention can rapidly provide high detection sensitivity and specificity, and does not require traditional bioenrichment (eg, enrichment culture), which is a surprising aspect, since all available methods require culture. In some embodiments, detection is possible within a single replication cycle of the phage, which is unexpected.

definition

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by the content, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in conjunction with, and references to, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein The teachings of immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods well known in the art and described in various general and more specific references discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein are those well known and commonly used in the art.

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

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

The use of the term "or" is used to mean "and/or" unless it is expressly stated that only alternatives or alternatives are mutually exclusive, although the disclosure supports a qualification that means only alternatives and "and/or". As used herein, "another" may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error of the device, method used to determine the value or the variation that exists between samples.

The term "solid support" or "support" means a structure that provides a matrix and/or surface on which biomolecules can be bound. For example, the solid support can be an assay well (i.e., such as a microtiter plate or multiwell plate), or the solid support can be a filter, an array, or a removable support, such as a bead or a position on a membrane (e.g., a filter plate or side stream).

The term "binding agent" refers to a molecule that can specifically and selectively bind a second (ie, different) target molecule. Interactions may be non-covalent, eg, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or may be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (ie, covalently or non-covalently bound) with a solid support.

As used herein, "analyte" refers to a molecule, compound or cell to be measured. In certain embodiments, an analyte of interest can interact with a binding agent. As used herein, the term "analyte" may refer to a protein or peptide of interest. Analytes can be agonists, antagonists or modulators. Alternatively, the analyte may not have a biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like.

The term "detectable moiety" or "detectable biomolecule" or "reporter protein" or "indicator" or "indicating moiety" refers to a molecule that can be measured in a quantitative assay. For example, an indicator moiety can include an enzyme that can be used to convert a substrate into a measurable product. The indicating moiety can be an enzyme (eg, luciferase) that catalyzes a reaction that produces a bioluminescent emission. Alternatively, the indicator moiety may be a quantifiable radioisotope. Alternatively, the indicating moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

As used herein, "bacteriophage" or "phage" includes one or more of a variety of bacterial viruses. In this disclosure, the terms "bacteriophage" and "phage" include, for example, mycobacteriophages (e.g., for TB or paraTB), mycophages (e.g., for fungi), mycoplasma phages, etc. Virus, and any other term referring to viruses that can invade live bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and use them to replicate themselves. Here, "microscopically visible" means that the largest dimension is one millimeter or less. Phages are viruses that have naturally evolved to use bacteria as their means of self-replication. The phage does this by attaching itself to the bacterium and injecting its DNA (or RNA) into the bacterium, and inducing it to replicate the phage hundreds or even thousands of times. Call this phage amplification.

As used herein, "late genetic region" refers to a region of the viral genome that is transcribed late in the viral life cycle. Late gene regions typically include the most abundantly expressed genes (eg, structural proteins that assemble into phage particles). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, late genes (synonymous with class III) are transcribed in phage T7, e.g., lipid cleavage from 8 min post infection, class I (e.g., RNA polymerase) are early from 4-8 min, and class II are early From 6-15 minutes, so there is an overlap in the timing of II and III. A late promoter is a promoter that is naturally located in such a late gene region and is active in this region.

As used herein, "enrichment culture" refers to traditional culture, such as incubation in a medium that favors the growth of microorganisms, and should not be confused with other possible uses of the word "enrichment", such as by removing the Enrichment of liquid components to concentrate the microorganisms contained therein, or other forms of enrichment that do not involve the traditional promotion of microbial reproduction. In some embodiments of the methods described herein, enrichment for very short periods of time may be used, but is not required and if used at all, the period of time is much shorter than traditional enrichment.

As used herein, "recombination" refers to a genetic (ie, nucleic acid) modification, usually performed in a laboratory, to assemble genetic material that would not otherwise be found. This term is used interchangeably with the term "modify" herein.

As used herein, "RLU" refers to the 96) or a relative unit of light measured by a similar instrument that detects light. For example, detection of a reaction between luciferase and a suitable substrate (e.g., using of ) are often reported as detected RLUs.

As used herein, "time to result" refers to the total time from sample preparation to data collection. The time to results does not include the time for any confirmatory testing.

sample

Embodiments of the methods and systems of the present invention, respectively, can allow rapid detection and quantification of microorganisms in a sample. For example, the method according to the invention can be carried out within a shortened period of time and with excellent results.

Microorganisms detected by the methods and systems of the present invention include pathogens of natural, commercial, medical or veterinary concern. Such pathogens include Gram-negative bacteria, Gram-positive bacteria, mycoplasma and viruses. Any microorganism for which an infectious agent specific for a particular microorganism has been identified can be detected by the method of the invention. Those skilled in the art will recognize that there are no limitations to the application of the methods of the invention other than the availability of the specific infectious agent/microbe pair required.

Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food or waterborne pathogens. Bacterial cells detectable by the present invention include, but are not limited to, all species of Salmonella, all strains of Escherichia coli, including but not limited to E. coli O157:H7, all species of Listeria, including but not limited to Not limited to Listeria monocytogenes (L. monocytogenes), and all species of Campylobacter. Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are pathogens of medical or veterinary importance. Such pathogens include, but are not limited to, Bacillus spp., Bordetella pertussis, Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens (Clostridium perfringens), Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Shigellasonnei , Staphylococcus aureus (Staphylococcus aureus) and several species of Streptococcus (Streptococcus spp.).

Samples can be environmental or food or water samples. Some embodiments may include medical or veterinary samples. Samples can be liquid, solid or semi-solid. A sample may be a swab of a solid surface. Samples may include environmental materials such as water samples, or filters from cyclone collectors for air samples or aerosol samples. Samples can be meat, poultry, processed foods, milk, cheese, or other dairy products. Medical or veterinary samples include, but are not limited to, blood, sputum, spinal fluid, and stool samples as well as different types of swabs.

In some embodiments, samples can be used directly in the detection methods of the invention without preparation, concentration or dilution. For example, liquid samples, including but not limited to, milk and juice, can be tested directly. Samples may be diluted or suspended in solutions, which may include, but are not limited to, buffers or bacterial culture media. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing or macerating the solid in the liquid. Samples should be maintained in a pH range that facilitates phage attachment to host bacterial cells. Samples should also contain suitable concentrations of divalent and monovalent cations, including but not limited to Na ⁺ , Mg ²⁺ and K ⁺ . Preferably, the sample is maintained at a temperature that maintains the viability of any pathogenic cells contained within the sample.

Preferably, in the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogenic cells present in the sample. During the step in which the phage attaches to the bacterial cells, the sample is preferably maintained at a temperature that promotes phage attachment. During the step in which the phage replicates within the infected bacterial cell or lyses the infected cell, the sample is preferably maintained at a temperature that promotes phage replication and host lysis. Such a temperature is at least about 25 degrees Celsius (°C), more preferably no greater than about 45°C. Most preferably about 37 degrees Celsius. It is also preferred to gently mix or shake the sample during phage attachment, replication and cell lysis.

Assays can include various suitable control samples. For example, a control sample without phage or a control sample containing phage but no bacteria can be assayed as a control for background signal levels.

indicator phage

As described in more detail herein, the compositions, methods, systems and kits of the invention may include infectious agents for the detection of pathogenic microorganisms. In certain embodiments, the invention includes recombinant indicator phages, wherein the phage genome has been genetically modified to include an indicator or reporter gene. In some embodiments, the invention may include compositions comprising a recombinant phage having an indicator gene incorporated into the phage genome.

The recombinant indicator phage may include a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during phage replication following infection of the host bacterium forms a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene region of the phage. Late genes are usually expressed at higher levels than other phage genes because they encode structural proteins. The late gene region may be a class III gene region and may include genes for major capsid proteins.

Some embodiments include designing (and optionally making) sequences for homologous recombination downstream of the major capsid protein gene. In some embodiments, the sequence comprises a codon-optimized reporter gene preceded by an untranslated region. Untranslated regions may include phage late gene promoters and ribosome entry sites.

In some embodiments, the indicator phage is derived from T7, T4, or other similar phage. The indicator phage can also be derived from T4-like, T7-like, ViI, ViI-like, CBA120 or combined with T7, T7-like, T4, T4-like, CBA120, ViI or ViI-like (or ViI virus-like according to GenBank/NCBI ) phage having 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, Another phage with a genome of 93, 94, 95, 96, 97, 98 or 99% homology. In some embodiments, the indicator phage is derived from a phage highly specific for a particular pathogenic microorganism. Genetic modification can avoid the deletion of wild-type genes, so the modified phage can remain more similar to the wild-type infectious agent than many commercially available phage. Environmentally derived phages may be more specific to bacteria found in the environment and thus be genetically distinct from commercially available phages.

Furthermore, phage genes considered non-essential may have unrecognized functions. For example, apparently non-essential genes may have important functions in elevated release, such as fine cutting, fitting, or tidying functions in assembly. Therefore, deleting a gene to insert an indicator can be deleterious. Most phages can package DNA several percent larger than their native genome. Given this consideration, a smaller indicator gene may be a more appropriate choice for modifying phages, especially those with smaller genomes. OpLuc and The protein is only about 20 kDa (about 500-600 bp to encode), while Fluc is about 62 kDa (about 1,700 bp to encode). In comparison, the genome of T7 is about 40 kbp, while that of T4 is about 170 kbp, and that of CBA120 is about 157 kbp. Furthermore, the reporter gene should not be expressed endogenously by the bacterium (i.e., not part of the bacterial genome), should yield a high signal-to-background ratio, and should be detectable in a timely manner. Promega is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, with Promega (imidazopyrazinone substrate (furimazine analog)) combination provides a robust signal with low background.

In some indicator phage embodiments, the indicator gene can be inserted into the untranslated region to avoid disruption of the functional gene, leaving the wild-type phage gene intact, which can lead to higher fitness when infecting non-laboratory strains of bacteria . Additionally, stop codons encompassing all three reading frames can increase expression by reducing readthrough, also known as leaky expression. This strategy can also eliminate the possibility of forming fusion proteins at low levels, which would appear as a background signal (eg, luciferase) that cannot be separated from phage.

Indicator genes can express various biomolecules. An indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes luciferase. Various types of luciferases can be used. In alternative embodiments, and as described in more detail herein, the luciferase is Oplophorus luciferase, luciferase luciferase, Lucia luciferase, Renilla luciferase, or an engineered One of the luciferases. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as

Thus, in some embodiments, the invention includes genetically modified phages that include a non-phage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. Use of viral late gene promoters ensures that the reporter gene (eg, luciferase) is not only expressed at high levels, like the viral capsid protein, but is not shut down, like endogenous bacterial genes or even early viral genes.

In some embodiments, the late promoter is a T4-, T7- or Vi1-like promoter, or another phage promoter similar (ie, without genetic modification) to that found in the selected wild-type phage. The late gene region may be a class III gene region and the phage may be derived from or have at least 70, 75, Another natural phage with a genome of 80, 85, 90 or 95% homology.

Genetic modification of infectious agents may include insertions, deletions, or substitutions of small fragments of nucleic acid, substantial portions of genes, or entire genes. In some embodiments, the inserted or replaced nucleic acid includes a non-native sequence. A non-native indicator gene can be inserted into the phage genome such that it is under the control of a phage promoter. In some embodiments, the non-native indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification can be configured such that the indicator protein product does not include a polypeptide of wild-type phage. In some embodiments, the indicator protein product is soluble. In some embodiments, the invention includes a method for detecting target bacteria comprising the step of incubating a test sample with such recombinant phage.

In some embodiments, expression of the indicator gene in progeny phage following infection of the host bacterium produces an episomal soluble protein product. In some embodiments, the non-native indicator gene is not adjacent to the gene encoding the structural phage protein, and thus no fusion protein is produced.

Unlike systems that use fusions of the detection moiety to the capsid protein (ie, fusion proteins), some embodiments of the invention express soluble luciferase. This can greatly increase the sensitivity of the assay (down to a single bacterium) and simplifies the assay, allowing for some embodiments to be completed in less than 1 hour, unlike the assays required due to the use of constructs that produce detectable fusion proteins. Several hours relative to other purification steps. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding constraints that may alter the conformation of the enzyme's active site or proximity to the substrate.

Furthermore, the number of moieties attached to protein subunits in the phage is limited by the defined fusion protein. For example, using a commercially available system designed as a platform for fusion proteins will result in about 415 copies of the fusion moiety, corresponding to about 415 copies of the gene 10B capsid protein per T7 phage particle. In the absence of this limitation, infecting bacteria can be expected to express more copies of the detection moiety (eg, luciferase) than are suitable on the phage. Additionally, large fusion proteins, such as capsid-luciferase fusions, may inhibit phage particle assembly and thus produce fewer phage progeny. Therefore, a soluble, non-fusion indicator gene product may be preferred.

In some embodiments, the indicator phage encodes a reporter protein, such as a detectable enzyme. The indicator gene product can produce 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 be used as indicator moieties. In some embodiments, luciferase is the indicating moiety. In some embodiments, Oplophorus luciferase is the indicating moiety. In some embodiments, an indicating moiety. Other engineered luciferases or other enzymes that produce a detectable signal may also be suitable indicator moieties.

In some embodiments, the use of a soluble detection moiety eliminates the need to remove contaminating parental phage from lysates of infected sample cells. Using the fusion protein system, any phage used to infect sample cells will have the detection moiety attached and will be indistinguishable from progeny phage that also contain the detection moiety. Because detection of sample bacteria relies on detection of newly formed (de novo) detection moieties, use of fusion constructs requires an additional step to separate the old (parental) moiety from the newly formed (progeny phage) moiety. This can be done by washing infected cells multiple times before the phage life cycle is complete, physically or chemically inactivating excess parental phage after infection, and/or chemically modifying parental phage with binding moieties such as biotin , which can then be bound and detached (eg by streptavidin-coated sepharose beads). However, even with all these attempts at removal, when high concentrations of parental phage are used to ensure infection of a small sample of cells, the parental phage may remain, creating background signals that may hinder phage detection from infected cell progeny.

In contrast, using an expressed soluble detection moiety in some embodiments of the invention, there is no need to purify the parental phage from the final lysate, since the parental phage will not have any attached detection moiety. Therefore, any detection moieties present after infection must reform to indicate the presence of infected bacteria. To take advantage of this benefit, production and preparation of the parental phage may involve purifying the phage from any episomal assays produced during the production of the parental phage in bacterial culture. According to some embodiments of the invention, phage can be purified using standard phage purification techniques such as sucrose density gradient centrifugation, cesium chloride isopycnic gradient centrifugation, HPLC, size exclusion chromatography and dialysis or derivatization techniques (such as Amicon band enrichment Device - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the recombinant phage preparation of the present invention to allow separation of parental phage particles from contaminating luciferase proteins produced upon phage propagation in bacterial hosts. In this way, the parental recombinant phage of the invention are substantially free of any luciferase produced during bacterial production. Removal of residual luciferase present in phage stocks can substantially reduce the background signal seen when recombinant phage are incubated with test samples.

In some embodiments of the modified phage, the late promoter (class III promoter, e.g., from T7, T4, or ViI) has high affinity for the RNA polymerase of the native phage transcribed for assembly into Genes for structural proteins in phage particles. These proteins are the most abundant proteins formed by phage, since each phage particle contains dozens or hundreds of copies of these molecules. Use of a viral late promoter ensures optimal high-level expression of the luciferase detection moiety. The initial wild-type phage specificity of the indicator phage was derived using a late viral promoter derived from the original wild-type phage from which the indicator phage was derived (e.g., a T4, T7, or ViI late promoter with a T4-, T7-, or ViI-based system) Late viral promoters (e.g., T4 or T7 late promoters with T4- or T7-based systems) or late viral promoters active under the original wild-type phage from which the indicator phage was derived (e.g., T4- or T7-based system-based T4 or T7 late promoter of the T7-system), which can further ensure optimal expression of the detection part. In some cases, the use of standard bacterial (non-viral/non-phage) promoters may be detrimental to expression, as these promoters are often downregulated during phage infection (for phage prioritization of bacterial sources for phage protein production ). Thus, in some embodiments, phage are preferably engineered to encode and express a soluble (episom) indicator moiety at high levels using substitutions in the genome that do not limit expression to the number of subunits of the phage structural components.

Compositions of the invention may comprise one or more wild-type or genetically modified infectious agents (eg, bacteriophage) and one or more indicator genes. In some embodiments, the composition may include a mixture of different indicator phages, which may encode and express the same or different indicator proteins.

Method for preparing indicator phage

An embodiment of a method for making an indicator phage begins with selection of a wild-type phage for genetic modification. Some phages are highly specific for target bacteria. This provides the opportunity for highly specific detection.

Thus, the methods of the invention take advantage of the high specificity of binding agents associated with infectious agents, which recognize and bind specific target microorganisms, as a means of amplifying the signal to detect low levels of microorganisms (e.g., single microorganisms) present in a sample . For example, infectious agents such as phages specifically recognize surface receptors of specific microorganisms, thereby specifically infecting these microorganisms. Therefore, these infectious agents may be suitable binders for targeting the microorganisms of interest.

A variety of infectious agents can be used. In alternative embodiments, bacteriophages, bacteriophages, mycobacteriophages (for example for TB and paraTB), mycophages (for example for fungi), mycoplasma phages and any other invasible live bacteria that can be used to target microorganisms of interest , fungi, mycoplasma, protozoa, yeast and other viruses of microscopic living organisms. For example, in one embodiment, when the target microorganism is a bacterium, the infectious agent can include a bacteriophage. For example, well-studied coliphages include T1, T2, T3, T4, T5, T7, and lambda; others available in the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1 . As discussed herein, phages can replicate within bacteria to produce hundreds of progeny phages. Detection of the product of the indicator gene inserted into the phage genome can be used as a measure of the bacteria in the sample.

Some embodiments of the present invention take advantage of the binding specificity and high-level genetic expression capabilities of recombinant phages for rapid and sensitive targeting to infect and facilitate detection of target bacteria. In some embodiments, the CBA120 phage is genetically modified to include a reporter gene. In some embodiments, the late gene region of the phage is genetically modified to include a reporter gene. In some embodiments, the reporter gene is located downstream of the major capsid gene. In other embodiments, the reporter gene is located upstream of the major capsid gene.

Some embodiments of the method for preparing a recombinant indicator phage include selecting a wild-type phage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid/vector comprising an indicator gene; transforming the homologous recombination plasmid/vector into a target pathogenic bacterium; using the selected wild-type phage Type phage infect transformed target pathogens, allowing homologous recombination between the plasmid/vector and phage genome; and isolate specific clones of recombinant phage.

Various methods for designing and preparing homologous recombination plasmids are known. Various methods are known for transforming bacteria with plasmids, including heat shock, F pili-mediated conjugation of bacteria, electroporation, and others. Various methods for isolating specific clones after homologous recombination are also known. Some method embodiments described herein use specific strategies.

Accordingly, some embodiments of the method of producing an indicator phage comprise the steps of: selecting a wild-type phage that specifically infects a target pathogen; determining the native sequence of the late region of the genome of the selected phage; annotating the genome and identifying the major capsid protein gene of the selected phage ; sequence designed for homologous recombination near the major capsid protein gene, wherein said sequence comprises a codon-optimized reporter gene; integration of sequence designed for homologous recombination into a plasmid/vector; transformation of the plasmid/vector to the target pathogen; select the transformed bacteria; infect the transformed bacteria with the selected wild-type phage to allow homologous recombination between the plasmid and the phage genome; determine the titer of the resulting recombinant phage lysate; and perform a limiting dilution assay to enrich Collection and isolation of recombinant phage. Some embodiments include further repeating the limiting dilution and titration steps after the first limiting dilution assay, as needed, until the recombinant phage represent a detectable portion of the mixture. For example, in some embodiments, the limiting dilution and titration steps can be repeated until at least 1/30 of the phage in the mixture are recombinant, and then specific recombinant phage clones are isolated. A weight:wild-type ratio of 1:30 is expected to yield an average of 3.2 transducing units (TU) per 96 plaques (eg, in a 96-well plate). With a Poisson distribution, the 1:30 ratio thus yields a 96% chance of seeing at least one TU somewhere in the 96 wells.

Figure 1 depicts a schematic representation of the wild-type CBA120 phage genome. Late gene clusters 110 were identified and open reading frames 120 (ORFs) in the late gene region were annotated. The ORF187/gp23 putative gene for major capsid protein 130 (MCP) was identified and its sequence was used together with the downstream sequence in the late gene cluster to generate a recombinant plasmid carrying the desired reporter gene.

Some embodiments of the method of making a recombinant indicator phage include designing a plasmid that can readily recombine with the wild-type phage genome to generate a recombinant genome. When designing the plasmid, some embodiments include adding a codon-optimized reporter gene, such as the luciferase gene. Some embodiments also include adding elements to the upstream untranslated region. For example, when designing a plasmid recombined with the CBA120 genome, the sequence encoding the C-terminus of the gp23/major capsid protein and The upstream untranslated region was added between the start codon of the reporter gene. Untranslated regions may include promoters, such as T4, T4-like, T7, T7-like, CBA120, ViI or ViI-like promoters. The untranslated region may also include a ribosome entry/binding site (RBS), also known as a "Shine-Dalgarno sequence" in bacterial systems. Either or both of these or other untranslated elements may be embedded within a short upstream untranslated region consisting of random sequences comprising about the same GC content as the rest of the phage genome. The random region should not include the ATG sequence as it will act as a start codon.

There are many known methods and commercial products for preparing plasmids. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be used in combination to prepare plasmids. Synthetic plasmids can also be ordered commercially (eg, GeneWiz). Cosmids can also be used, or the CRISPR/Cas9 system can be used to selectively edit the phage genome.

Figure 2 shows an embodiment of a plasmid designed for recombination with the CBA120 phage genome to generate recombinant phage. This particular plasmid was named pUC57.HR.CBA120.NanoLuc. The detection/indication part consists of Reporter gene 941-1540 encoding. The insert (396-1883) is the canonical AmpR form of pUC57. The major capsid protein C-terminal fragment is represented by 396-895, ORF187/gp23. The T4-like phage late promoter consensus sequence (902-912) and the Shine-Dalgarno ribosome entry/binding site (927-934) within the 5' untranslated region are represented by 896-940. codon optimized Reporter genes are indicated by 941-1540. The untranslated region (UTR) and N-terminal fragment of ORF185 putative protein are indicated by 1541-1838. The transcription terminator (1839-1883) was only in the plasmid and was not part of the phage genome due to recombination.

ORF187/gp23 fragment 396-895 is part of the structural gene encoding virion proteins. Since these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels as long as the late gene promoter and/or other similar control elements are used.

Figure 3 shows a schematic diagram of the expected homologous recombination between the plasmid of Figure 2 and the phage genome of Figure 1 to generate recombinant phage expressing the luciferase gene. In this embodiment of homologous recombination to generate recombinant phage, the CBA120 phage genome is 157,304 base pairs, while the synthetic plasmid is 4,117 base pairs. The final recombinant genome resulting from recombination was 157,949 base pairs.

In some embodiments, the indicator phage according to the present invention comprises a genetically engineered CBA120 phage comprising a reporter gene, such as a luciferase gene. For example, the indicator phage can be CBA120 phage, where the genome contains the sequence of the gene. The recombinant CBA120 phage genome may further comprise T4, T7, CBA120, ViI or another late promoter.

Thus, in the embodiment of the recombinant phage produced due to recombination as shown in Figure 3, the indicator gene (i.e., ) is inserted into the late gene region just downstream of the gene encoding the major capsid protein, and thus produces a Genetic recombinant phage genome. The construct may additionally comprise a consensus T4, T7, CBA120, ViI or another late promoter or another suitable promoter to drive transcription and expression of the luciferase gene. The construct may also contain a composite untranslated region synthesized from several UTRs. This construct ensures the production of soluble luciferase so that expression is not limited to the amount of capsid protein inherent in the phage display system.

Figure 4 depicts the isolation of recombinant phage from a mixture of wild-type and recombinant phage produced by homologous recombination shown in Figure 3 using the plasmid constructs shown in Figure 2.

In a first step 402, bacteria transformed with homologous recombination plasmids are infected with phages to form progeny phages having a mixture of parental and recombinant phages in a ratio of approximately 120 wild-type 432:1 recombinant phages 434. The resulting recombinant phage mixture was diluted 404 into a 96-well plate 406 to obtain an average of 3 recombinant transducing units (TU) per plate, which corresponds to approximately 3.8 infectious units (IU) per well for most wild-type phage. ). The 96-well plates were assayed for luciferase activity to identify wells 436 containing recombinant phage compared to wells 440 containing wild-type phage. Add 408 bacteria 438; for example, each well may contain approximately 50 μL of a turbid E. coli O157:H7 culture. This allows the phage to replicate and produce luciferase442. After incubation for 2 hours at 37°C as indicated at 410, wells can be screened for the presence of luciferase 442. Any positive wells will likely have been inoculated with a single recombinant phage, and at this stage the mixture can contain a ratio of approximately 3.8 wild-type phage:1 recombinant, an enrichment above the initial 120:1 ratio. In one embodiment, soluble luciferase and phage are present in a ratio of about 16 wild-type:1 recombinant. If desired (i.e., if the recombinant:wild type ratio is below 1:30), progeny from this enrichment culture 412 may undergo additional limiting dilution assays 414 to increase the ratio and determine the transduced units of recombinant phage actual concentration. For example, about 3 recombinant TU/96-well plates 416 can be aliquoted 414 from the first purification stock to form a second dilution test plate 420 inoculated with about ~20 mostly wild-type phage/well. Any positive luciferase wells would likely have been inoculated with a single recombinant along with -20 wild-type phage. These wells can be assayed for the presence of luciferase 442.

After addition of bacteria and incubation (eg, 2 hours at 37°C) 418 soluble luciferase and phage are present in approximately 20 wild-type:1 recombinant 420 . Finally, a plaque assay 422 can be performed to screen for recombinants expressing luciferase 446 . A small number of individual (eg, n=48) plaques can be individually picked and screened 426 for luciferase activity 436 in a third multi-well plate. In one embodiment, this method should ensure that about 3 recombinants are in the plaque mixture to be screened. One plaque can be removed from the plate into each well 424 of the 96-well plate and a luciferase assay 426 performed to determine which well contains phage exhibiting luciferase 442 activity. Wells 428 demonstrating luciferase activity represent pure recombinant phage 434 , while wells 430 without luciferase activity represent pure wild-type phage 432 .

Individual plaques can then be suspended in buffer (e.g., 100 μL TMS) or medium, and aliquots (e.g., about 5 μL) added to wells containing turbid E. coli O157:H7 cultures, and assayed after incubation (eg, about 45 minutes to 1 hour at 37°C). Positive wells are expected to contain pure recombinant phage cultures. Certain embodiments may include another round of plaque purification.

Thus, as illustrated in Figure 4, recombinant phage produced by homologous recombination of plasmids designed for recombination with the wild-type phage genome can be isolated from a mixture constituting only 0.005% of the total phage genome. After isolation, large-scale production can be performed to obtain high-titer recombinant indicator phage stocks suitable for use in E. coli O157:H7 detection assays. Additionally, cesium chloride isopycnic centrifugation can be used to separate phage particles from contaminating luciferin proteins to reduce background.

5 shows an electron micrograph of one embodiment of a recombinant indicator phage as shown in FIG. 3 produced by recombination of the wild-type CBA120 phage genome shown in FIG. 1 with the plasmid shown in FIG. 2 . To capture images, phage were purified on a 5–20% sucrose density gradient, adsorbed on glow-discharge-treated carbon membranes, and stained with 2% uranyl acetate. Samples were observed in a FEI Tecnai G ² Spirit BioTwin transmission electron microscope, and micrographs were taken with an Eagle ^TM 2KCCD. This indicator phage was named "CBA120NanoLuc" (or "CBA120NanoLuc indicator phage") and was used in the assays described herein. The data presented in the Examples and Figures herein were obtained using the indicator phage used to infect bacteria in the test samples.

In this way, and as described in more detail in the Examples below, a reporter gene of interest (e.g., a luciferase gene such as Firefly, Oplophorus, or an engineered luciferase such as ) recombinant phage.

Method for detecting microorganisms using infectious agents

As described herein, in certain embodiments, the invention may include methods of detecting microorganisms using infectious particles. The method of the invention can be implemented in various ways.

In one embodiment, the present invention may include a method for detecting a target bacterium in a sample, comprising the step of incubating the sample with a phage that infects the target bacterium, wherein the phage contains an indicator gene that allows the phage to replicate after infecting the target bacterium Expression of the indicator gene during the process produces a soluble indicator protein product; detection of the indicator protein product wherein positive detection of the indicator protein product indicates the presence of the target bacteria in the sample.

In certain embodiments, assays can be performed to utilize general concepts that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using recombinant phages of the present invention (i.e., indicator phages) may allow rapid detection of specific bacterial strains with total assay times between 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, Within 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12 hours, depending on sample type, sample size, and assay format. For example, depending on the phage strain and bacterial strain to be detected in the assay, the type and size of the sample to be tested, the conditions required for the survival of the target, the complexity of the physical/chemical environment, and the concentration of "endogenous" non-target bacterial contaminants , the required time can be shorter or longer.

Figure 6 illustrates an embodiment of an assay for detection of target bacteria using modified phage according to one embodiment of the present invention. Aliquots of indicator phage 614 are dispensed into individual wells 602 of multiwell plate 604, and test sample aliquots containing bacteria 612 are then added and incubated (606) for a period of time (e.g., 45 minutes at 37°C) such that Sufficient to replicate phage and produce soluble indicator 616 (eg, luciferase). The plate wells 608 containing the soluble indicator and phage can then be assayed (610) to measure indicator activity on the plate 618 (eg, luciferase assay). This paper describes experiments utilizing this method. In some embodiments, test samples are not concentrated (eg, by centrifugation), but are directly incubated with indicator phage for a period of time prior to assaying for luciferase activity. In other embodiments, samples can be concentrated prior to enrichment or prior to testing using various means (eg, centrifuges or filters). For example, 10 mL aliquots of prepared samples can be extracted and centrifuged to pellet cells and large debris. The pellet can be resuspended in a smaller volume for enrichment or for testing (ie, prior to infection of the sample with the indicator phage).

In some embodiments, samples can be enriched by incubation under growth-promoting conditions prior to testing. In such embodiments, the enrichment time may be 1, 2, 3, 4, 5, 6, 7 or up to 8 hours or longer depending on the sample type and size.

Thus, in some embodiments, an indicator phage includes a detectable indicator moiety, and infection of a single pathogen cell (eg, bacterium) can be detected by a signal generated by the enlarged indicator moiety. Accordingly, the method may comprise detecting an indicator moiety produced during phage replication, wherein detection of the indicator indicates the presence of the target bacterium in the sample.

In one embodiment, the present invention may include a method for detecting target bacteria in a sample, comprising the steps of: incubating the sample with a recombinant phage that infects the target bacteria, wherein the recombinant phage includes an indicator gene inserted into the late gene region of the phage, such that expression of the indicator gene during phage replication following infection of the host bacterium results in the production of a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the amount of the indicating moiety detected corresponds to the amount of target bacteria present in the sample.

As described in more detail herein, the methods and systems of the invention can utilize a range of concentrations of a parental indicator phage to infect bacteria present in a sample. In some embodiments, the indicator phage is added to the sample at a concentration sufficient to rapidly find, bind and infect very small numbers of target bacteria (eg, single cells) present in the sample. In some embodiments, the phage concentration may be sufficient to find, bind and infect target bacteria in less than an hour. In other embodiments, these events may occur in less than 2 hours or less than 3 hours after the addition of the indicator phage to the sample. For example, in certain embodiments, the incubation step has a phage concentration greater than 1 x 10 ⁵ PFU/mL, greater than 1 x 10 ⁶ PFU/mL, or greater than 1 x 10 ⁷ PFU/mL.

In certain embodiments, the recombinant infectious agent can be purified so as to be free of any residual indicator protein produced when the infectious agent stock was produced. Thus, in certain embodiments, recombinant phage can be purified using cesium chloride isopycnic gradient centrifugation prior to incubation with the sample. Where the infectious agent is a phage, this purification may have the added benefit of removing phage that do not have DNA (ie, empty phage or "ghost cells").

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

Aliquots of the test sample can be dispensed directly into wells of a multiwell plate, indicator phage can be added, and after a period sufficient for infection, lysis buffer can be added along with a substrate for the indicator moiety (e.g., for fluorescent Luciferase substrate for luciferase indicator) and assay for detection of indicator signal. Some embodiments of the method can be performed on filter plates. Some embodiments of the method can be performed with or without concentrating the sample prior to infection with the indicator phage.

For example, in many embodiments, assays are performed using multiwell plates. The choice of plate (or any other container in which the assay can be performed) can affect the assay procedure. For example, some flat panels may include colored or white backgrounds, which may affect the detection of light emissions. In general, white plates have higher sensitivity, but also produce higher background signal. Plates of other colors may produce lower background signal, but also slightly less sensitivity. Additionally, one cause of background signal is light leakage from one well to another adjacent well. There are some plates with white holes, but the rest of the plates are black. This allows high signal within the well but prevents well-to-well light leakage and thus can reduce background. Therefore, the choice of plate or other assay container may affect the sensitivity and background signal of the assay.

The methods of the invention may include various other steps to increase sensitivity. For example, as discussed in more detail herein, the method may include a step of washing the captured and infected bacteria after addition of phage but prior to incubation to remove excess parental phage and/or luciferase or other reports of contaminating phage preparations protein.

In some embodiments, detection of target microorganisms can be accomplished without culturing the sample to increase the population of microorganisms. For example, in certain embodiments, the total time required for detection is less than 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 Hours, 1.5 hours, 1.0 hours, 45 minutes or less than 30 minutes. Minimizing time to results is critical for food and environmental testing for pathogens.

In contrast to assays known in the art, the methods of the present invention can detect individual microorganisms. Thus, in certain embodiments, the method can detect < 10 microbial cells (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms) present in a sample. For example, in certain embodiments, the recombinant phage is highly specific for E. coli O157:H7. In one embodiment, the recombinant phage can differentiate E. coli O157:H7 in the presence of more than 100 other types of bacteria. In certain embodiments, recombinant phage can be used to detect specific types of individual bacteria in a sample. In certain embodiments, 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.

Accordingly, various aspects of the invention provide methods for determining microorganisms in a sample by detection of an indicator moiety. In some embodiments, where the target microorganism is a bacterium, the indicator moiety can be associated with an infectious agent (eg, an indicator phage). The indicator moiety can react with a substrate to emit a detectable signal or can emit an intrinsic signal (eg, a fluorescent protein). In some embodiments, the detection sensitivity can reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 target microbial cells in the test sample. In some embodiments, even a single cell of the microorganism of interest can produce a detectable signal. In some embodiments, the phage is a T4-like or ViI-like phage. In some embodiments, the recombinant phage is derived from CBA120. In certain embodiments, the CBA120 recombinant phage is highly specific for E. coli O157:H7.

In some embodiments, the indicator moiety encoded by the infectious agent can be detected during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicating moieties are known in the art, and many are commercially available. In some embodiments, the indicator phage includes an enzyme, which serves as the indicator moiety. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. Indicators can emit 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 be used as indicator moieties. In some embodiments, luciferase is the indicating moiety. In some embodiments, Oplophorus luciferase is the indicating moiety. In some embodiments, is the instruction part. Other engineered luciferases or other enzymes that produce a detectable signal are also suitable indicator moieties.

Thus, in some embodiments, the recombinant phage of the method, system or kit is produced from wild-type phage CBA120. In some embodiments, the indicator gene encodes a protein that emits an intrinsic signal, such as a fluorescent protein (eg, green fluorescent protein or otherwise). Indicators may emit light and/or may be detected by a color change. In some embodiments, the indicator gene encodes an enzyme (eg, luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of: Oplophorus luciferase, Firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase or engineered Luciferase as Rluc8.6-535 or Orange Nano-lantern.

Detecting the indicator can include detecting the emission of light. In some embodiments, a luminometer can be used to detect the reaction of an indicator (eg, luciferase) with a substrate. Detection of RLU can be achieved with a luminometer, or other instruments or devices can be used. For example, a spectrophotometer, CCD camera, or CMOS camera can detect color changes and other light emissions. Absolute RLU is important for detection, but the signal-to-background ratio also needs to be high (eg, >2.0, >2.5, or >3.0) for reliable detection of single cells or small numbers of cells.

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

Accordingly, in some embodiments utilizing indicator phages, the present invention includes methods for detecting target microorganisms comprising the steps of: capturing at least one sample bacterium; incubating at least one bacterium with a plurality of indicator phages; allowing infection and replication of a period of time to produce progeny phage and express the soluble indicator moiety; detection of progeny phage, or preferably the indicator, wherein detection of the indicator demonstrates the presence of bacteria in the sample.

For example, in some embodiments, test sample bacteria can be captured by binding to the surface of a plate, or by filtering the sample through a bacterial filter (eg, a 0.45 μm pore size spin filter or plate filter). In one embodiment, an infectious agent (eg, an indicator phage) is added in minimal volume to the sample captured directly on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are subsequently washed one or more times to remove excess unbound infectious agent. In one embodiment, culture medium (e.g., Luria-Bertani Broth, also referred to herein as LB, or Tryptic Soy Broth or Tryptone Soy Broth, also referred to herein as TSB) may be added to further Incubation was performed for a period of time to allow replication of bacterial cells and phage and high level expression of the genes encoding the indicated portions. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage only needs to be long enough for a single phage life cycle. It was previously thought that more time would be required to use the amplification capabilities of the phage, so that the phage would replicate for several cycles. According to some embodiments of the invention, a single replication cycle of the indicator phage may be sufficient to facilitate sensitive and rapid detection.

In some embodiments, after applying an aliquot of a test sample containing bacteria to a spin column and infection with recombinant phage and optional washing to remove any excess phage, the level of soluble indicator detected will be compared to that produced by the infected bacteria. Phage content is proportional.

When the bacteria lyse, a soluble indicator (eg, luciferase) is released into the surrounding fluid, which can then be measured and quantified. In one embodiment, the solution is spun through a filter, and after addition of a substrate for the indicator enzyme (e.g., a luciferase substrate), the solution is collected for testing in a new container (e.g., in a luminometer). filtrate. Alternatively, the indicator signal can be measured directly on the filter.

In various embodiments, the purified parental indicator phage does not include the detectable indicator itself, since the parental phage can be purified prior to use in incubation with a test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the invention, the parental phage can be purified to exclude any existing indicator protein (eg, luciferase). In some embodiments, expression of the indicator gene during phage replication following infection of the host bacterium forms a soluble indicator protein product. Thus, in many embodiments, it is not necessary to separate the parents from the progeny phage prior to the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a phage. In one embodiment, the indicator moiety is a soluble luciferase that is released upon lysis of the host microorganism.

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

For example, in one embodiment, the invention may include a method for detecting E. coli O157:H7 comprising the step of infecting a 96-well filter plate with multiple parental indicator phages capable of expressing luciferase upon infection. cells; by washing off excess phage, adding LB broth and allowing time for the phage to replicate and lyse specific E. coli targets (eg, 30-90 minutes); and by adding luciferase substrate and directly in 96-well plates Indicator luciferase is detected by measuring luciferase activity, wherein detection of luciferase activity indicates the presence of E. coli O157:H7 in the sample.

In another embodiment, the present invention may include a method for detecting Escherichia coli O157:H7 comprising the step of: infecting a liquid solution in a 96-well plate or cells in suspension; time allowed for phage to replicate and lyse specific E. coli targets (e.g., 30-120 minutes); and determination of luciferase activity by adding luciferase substrate and measuring luciferase activity directly in 96-well plates Detection indicator luciferase, wherein detection of luciferase activity indicates the presence of E. coli O157:H7 in the sample. In such embodiments, no capture step is required. 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/tryptone soy broth, peptone water, or nutrient broth. In some embodiments, the liquid solution or suspension may be bacteria diluted in LB broth.

In some embodiments, lysis of bacteria can occur before, during or after the detection step. Experiments show that, in some embodiments, infected unlysed cells are detectable upon addition of a luciferase substrate. It is speculated that the luciferase may have escaped from the cells and/or the luciferase substrate may have entered the cells without complete cell lysis. Thus, for embodiments utilizing a spin filter system, where only luciferase released into the lysate (rather than luciferase still inside intact bacteria) is analyzed in the luminometer, lysis is required for detection. However, for embodiments utilizing filter plates or 96-well plates and samples in solution or suspension, where the initial plate filled with intact and lysed cells is assayed directly in the luminometer, no lysis is required for detection.

In some embodiments, the reaction of the indicating moiety (eg, luciferase) with the substrate may last for 30 minutes or longer, and detection at different time points may be desirable for optimal sensitivity. For example, in an embodiment using a 96-well filter plate as the solid support and luciferase as the indicator, photometer readings can be taken initially and at 10- or 15-minute intervals until the reaction is complete.

Surprisingly, the use of high concentrations of phage for infection of test samples successfully achieved the detection of very small numbers of target microorganisms in a very short period of time. In some embodiments, the phage need only be incubated with the test sample for a length of time sufficient for the life cycle of a single phage. In some embodiments, the phage concentration used for this incubation step is higher than 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ , 1.2×10 ⁷ , 1.3×10 ⁷ , 1.4×10 ⁷ , 1.5×10 ⁷ , 1.6×10 ⁷ , 1.7×10 ⁷ , 1.8×10 ⁷ , 1.9×10 ⁷ , 2.0×10 ⁷ , 3.0×10 ⁷ , 4.0×10 ⁷ , 5.0×10 ⁷ , 6.0×10 ⁷ , 7.0×10 ⁷ , 8.0×10 ⁷ , 9.0×10 ⁷ or 1.0×10 ⁸ PFU/mL.

The success of using such high concentrations of phage is surprising since large numbers of phage have previously been associated with "lysis from the outside", which kills target cells and thus prevents the generation of useful signals from earlier phage tests. It may be that the cleanup of prepared phage stocks as described herein helps alleviate this problem (for example, by cesium chloride isopycnic gradient ultracentrifugation), because in addition to removing any contaminating luciferase associated with the phage, This clearance also likely removed decidual particles (particles with lost DNA). This slender particle can lyse the bacterial cell by "lysis from the outside", killing the cell prematurely and thereby preventing the generation of the indicator signal. Electron microscopy demonstrated that crude phage lysates (ie, prior to cesium chloride cleanup) had greater than 50% sloughs. These decidual particles can cause premature death of microorganisms through the action of many phage particles that penetrate the cell membrane. Thus, slender particles may cause previous problems where high PFU concentrations were reported to be detrimental. Furthermore, very clean phage preparations allow testing without washing steps, which allows assays to be performed without an initial concentration step. Some embodiments do include an initial concentration step, and in some embodiments, this concentration step allows for shorter enrichment incubation times.

Some embodiments of the test methods may further include validation assays. Various assays are known in the art to confirm initial results, usually at a later time point. For example, samples can be cultured (for example, as described in Example 4 / Assays), PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm initial results.

Figures 7-9 show data from a basic assay (eg, performed as shown in Figure 6) using the CBA120NanoLuc indicator phage on samples from E. coli O157:H7 cultures. Figure 7 shows three different infectious phage concentrations, 10 ⁵ , 10 ⁶ and 10 ⁷ phage/mL. Figure 8 uses 6-10 replicates for each indicated cell number to demonstrate significant differences between signals from single cells compared to zero cells (background) or higher numbers of cells. Figure 9 shows that the signal to background ratio for the experiment shown in Figure 8 was greater than 2.0. Example 3 also describes these experiments.

Beef Determination

Existing protocols for detecting E. coli O157:H7 in food are complex, expensive, slow, labor intensive and prone to false positives. Detection with recombinant phage specific for this pathogen provides an efficient, rapid and simple alternative to testing.

Embodiments of the beef assay include a sample preparation step. Some embodiments may include an enrichment time. For example, depending on sample type and size, 1, 2, 3, 4, 5, 6, 7 or 8 hours of enrichment may be required. Following these sample preparation steps, infection with high concentrations of recombinant phage expressing reporters or indicators can be performed in a variety of assay formats, such as shown in FIG. 6 .

Embodiments of the beef assay can detect individual pathogenic bacteria in sample sizes corresponding to industry standards, with a 20-50% reduction in time to result, depending on sample type and size.

Figures 10-16 show data from beef assay experiments using CBA120NanoLuc indicator phage, as described in Example 4.

Vegetable Wash Analysis

To prepare a vegetable wash, you can weigh vegetable leaves (such as spinach or lettuce) and add them to a clean plastic bag. The liquid can be added to the vegetable wash. For example, in some embodiments, 5 mL of water is added per gram (g) of vegetables. Other laboratory fluids (eg LB) can also be used. The leaves and solution can be mixed by hand for a few minutes. The liquid can then be extracted from the plastic bag and can be used as "vegetable wash". Using this method, approximately 1 million "endogenous" bacterial contaminants were found to be present on a single spinach leaf (1-2 g).

The assay is quantitative because the detected signal is proportional to the amount of target bacteria in the sample. For example, a known number of E. coli O157:H7 cells can be added to a vegetable wash sample to simulate vegetable contamination with pathogenic bacteria. Experiments using the vegetable wash samples described in Example 5 demonstrated significant differences between the signals from 0 cells, 1 cell, and 7 cells per assay, demonstrating the ability to detect single digit cell numbers in vegetable washes. Using more bacterial cells per assay showed increased signal in a dose-dependent manner. The vegetable wash contained approximately ¹⁰⁶ non-target bacteria/mL, corresponding to at least 105 non ^- target bacteria per sample in this assay (including the 0-cell E. coli O157:H7 control). ^The ability to discern as few as a single target bacterial cell from 105 non-target bacteria was surprising and again demonstrated the specificity and sensitivity of the assay. Figure 17 shows data from a vegetable washing experiment (Example 5).

In some embodiments, the incubation step of the methods described herein comprises a final phage concentration of greater than 7x 10 ⁶ , 8x 10 ⁶ , 9x 10 ⁶ , 1.0x 10 ⁷ , 1.1x 10 ⁷ , 1.2x 10 ⁷ , 1.3x 10 ⁷ , 1.4x 10 ⁷ ,1.5x 10 ⁷ ,1.6x 10 ⁷ ,1.7x 10 ⁷ ,1.8x 10 ⁷ ,1.9x 10 ⁷ ,2.0x 10 ⁷ ,3.0x 10 ⁷ ,4.0x 10 ⁷ ,5.0x 10 ⁷ , 6.0x 10 ⁷ , 7.0x 10 ⁷ , 8.0x 10 ⁷ , 9.0x 10 ⁷ , or 1.0x 10 ⁸ PFU/mL. Such high phage concentrations were previously reported to be detrimental to this assay, so the successful use of such high concentrations produced unexpected results. In some embodiments, the methods of the invention require less than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours to detect the target microorganism. In some embodiments, the method detects as few as 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 target bacterial cells. These time frames are shorter than previously thought. In some embodiments, even single cells of bacteria are detectable. In additional embodiments, the invention includes systems (eg, computer systems, automated systems, or kits) comprising components for performing the methods disclosed herein, and/or using the modified phage described herein.

Systems and kits of the invention

In some embodiments, the invention includes systems (eg, automated systems or kits) that include components for performing the methods disclosed herein. In some embodiments, an indicator phage is comprised in a system or kit according to the invention. The methods described herein can also use such indicator phage systems or kits. Given the small quantities of reagents and materials required to perform the methods, some embodiments described herein are particularly suited for automation and/or kits. In certain embodiments, each component of the kit can comprise a self-contained unit that can be transferred from a first site to a second site.

In some embodiments, the invention includes systems or kits for the rapid detection of target microorganisms in a sample. In certain embodiments, the system or kit includes a component for incubating a sample with an infectious agent specific for a microorganism of interest and a component for detecting an indicator moiety, wherein the infectious agent includes the indicator moiety. In some embodiments of the systems and kits of the present invention, the infectious agent is a recombinant phage that infects the target bacterium, and the recombinant phage contains an indicator gene inserted into the late gene region of the phage as an indicator moiety, allowing the phage to replicate after infection of the host bacterium Expression of the indicator gene during the process forms a soluble indicator protein product. Some systems further include components for capturing target microorganisms on a solid support.

In other embodiments, the present invention includes a method, system or kit for the rapid detection of a target microorganism in a sample comprising an infectious agent component specific for the target microorganism, wherein the infectious agent comprises an indicator moiety, and for Detect the components of the indicator section. In some embodiments, the phage is a T4-like, ViI, ViI-like or CBA120 phage. In one embodiment, the recombinant phage is derived from CBA120. In certain embodiments, recombinant phage are highly specific for a particular bacterium. For example, in certain embodiments, the recombinant phage is highly specific for E. coli O157:H7. In one embodiment, the recombinant phage can differentiate E. coli O157:H7 in the presence of more than 100 other types of bacteria. In certain embodiments, a system or kit detects a particular type of individual bacteria in a sample. In certain embodiments, the system or kit detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 specific bacteria.

In certain embodiments, the system and/or kit can further include components for washing the captured microbial sample. Additionally or alternatively, the system and/or kit may further comprise a component for determining the amount of the indicator moiety, wherein the detected amount of the indicator moiety corresponds to the amount of the microorganism in the sample. For example, in certain embodiments, a system or kit can include a luminometer or other device for measuring luciferase activity.

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

Accordingly, in certain embodiments, the present invention may include a system or kit for the rapid detection of a target microorganism in a sample comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein The infectious agent includes an indicator moiety; a moiety for capturing microorganisms from a sample on a solid support; a moiety for washing the captured microbial sample to remove unbound infectious agent; and a moiety for detecting the indicator moiety. In some embodiments, the same components can be used for the steps of capture and/or incubation and/or washing (eg, filter modules). Some embodiments additionally comprise a moiety for determining the amount of a target microorganism in a sample, wherein the detected amount of the indicating moiety corresponds to the amount of the microorganism in the sample. Such a system may include various embodiments and sub-embodiments similar to those described above for the rapid method of detecting microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage. In a computerized system, the system can be fully automated, semi-automated, or computer-guided by the user (or some combination thereof).

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

In one embodiment, the invention includes a system or kit comprising a composition for detecting a microorganism of interest, comprising: a composition for isolating the microorganism of interest from other components in a sample; infecting at least one component of the microorganism with multiple parental infectious agents; for lysing at least one infected microorganism to release the components of the progeny infectious agent present in the microorganism; and for detecting the progeny infectious agent or (with greater sensitivity) Detecting a fraction of a soluble protein encoded and expressed by an infectious agent, wherein detection of the infectious agent or soluble protein product of the infectious agent indicates the presence of the microorganism in the sample. Infectious agents may include CBA120NanoLuc.

A system or kit can include various components for detecting progeny infectious agents. For example, in one embodiment, a progeny infectious agent (eg, phage) can include an indicator moiety. In one embodiment, the indicator moiety in the progeny infectious agent (eg, phage) can be a detectable moiety expressed during replication, such as a soluble luciferase protein.

In other embodiments, the invention may include a kit for the rapid detection of a target microorganism in a sample, the system comprising: components for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent includes an indicator a moiety; a moiety for capturing microorganisms from a sample on a solid support; a moiety for washing the captured microbe sample to remove unbound infectious agents; and a moiety for detecting the indicator moiety. In some embodiments, the same components can be used for the capture and/or incubation and/or washing steps. Some embodiments additionally include a moiety that is expanded to determine the amount of microorganisms of interest in a sample, wherein the detected amount of the indicative moiety corresponds to the amount of microorganisms in the sample. Such kits may comprise various embodiments and sub-embodiments similar to those described above for the method of rapid detection of microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

In some embodiments, a kit can include components for isolating a microorganism of interest from other components in a sample.

The systems and kits of the invention include various components. As used herein, the term "component" is defined broadly and includes any suitable device or collection of devices suitable for performing the method. The component parts need not be completely connected or positioned relative to each other in any particular way. The invention includes any suitable arrangement of the components relative to each other. For example, components do not need to be in the same room. In some embodiments, however, the components are connected to each other in a complete set. In some embodiments, the same component can perform multiple functions.

Computer system and computer readable medium

The system described in the invention or any part thereof may be embodied in the form of a computer system. Typical examples of computer systems include general-purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit components, and other devices or arrangements of devices capable of performing the steps making up the methods of the present technology.

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 can be connected to the communication bus. A computer may also include memory. The memory may include random access memory (RAM) and read only memory (ROM). The computer system may further include storage devices. The storage device can be a hard drive or a removable storage device such as a floppy drive, optical drive, etc. The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet through the I/O interface. The communication unit allows data to be transferred to and received from other databases. The communication unit may include a modem, Ethernet card or any similar device that enables the computer system to connect to databases and networks such as LAN, MAN, WAN and the Internet. The computer system thus facilitates input from the user through the input device, through the I/O interface into the system.

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

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

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

Such devices may also include computer readable storage medium readers, communication devices (eg, modems, network cards (wireless or wired), infrared communication devices, etc.) and working memory as described above. A computer-readable storage medium reader may be connected to or configured to receive a computer-readable storage medium representing remote, local, fixed and/or removable storage and for temporary and/or More storage media permanently containing, storing, transmitting and receiving computer readable information. The system and various devices will also typically include various software applications, modules, servers or other elements located within at least one working storage device, including operating systems and application programs, such as client applications or web browsers. It should be appreciated that alternative embodiments may have variations from those described above. For example, custom hardware may also be used and/or specific elements may be implemented in hardware, software (including portable software, such as applets), or both. Additionally, connections to other computing devices such as network input/output devices may be used.

Non-transitory storage media and computer readable media for containing code or a portion of code may include any suitable medium, including storage media and communication media, known or used in the art, such as but not limited to, implementing in any method or technology volatile and nonvolatile, removable and non-removable media for the storage and/or transmission of information (such as computer readable instructions, data structures, program modules, or other data), including RAM, ROM, EEPROM, flash memory or other storage technology, CD-ROM, digital versatile disc (DVD) or other optical storage, magnetic cartridges, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other Take the medium. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will recognize other ways and/or methods for implementing the various embodiments.

A computer readable medium may include, but is not limited to, an electrical, optical, magnetic, or other storage device capable of providing computer readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROM, RAM, SRAM, DRAM, content addressable memory ("CAM"), DDR, flash memory such as NAND flash or NOR flash , ASIC, configured processor, optical storage, tape or other magnetic storage, or any other medium from which a computing 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 cache memory. The computing device can communicate with one or more external computer-readable media, such as an external hard drive or an external DVD or Blu-Ray drive.

As discussed above, the embodiments include a processor configured to execute computer-readable program instructions and/or access information stored in memory. Instructions may include processor-specific instructions generated by an assembler 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, a computing device includes a single processor. In other embodiments, a device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronics such as PLCs, Programmable Interrupt Controllers (PICs), Programmable Logic Devices (PLDs), Programmable Read-Only Memory (PROM), Electrically Programmable Read-Only Memory (EPROM) or EEPROM), or other similar devices.

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

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

The set of instructions for execution by a computer system may include various requirements for instructing a processor to perform a particular task, such as steps making up a method of the present technology. The set of instructions may be in the form of a software program. Furthermore, software may be in the form of a collection of separate programs, a program module with a larger program, or a portion of a program module, as in the techniques herein. Software may also include modular programming in object-oriented programming. The processing of input data by a processor may be in response to a user request, the result of previous processing, or a request made by another processor.

Although the invention has been disclosed with respect to certain embodiments, various modifications, changes and variations of the described embodiments are possible without departing from the scope and spirit of the invention as defined in the appended claims. Accordingly, the invention is considered not to be limited to the described embodiments, but to have its full scope defined by the language of the following claims and equivalents thereof.

Some embodiments of the technology described herein may be defined according to any of the following numbered paragraphs:

(1) A recombinant phage comprising an indicator gene inserted into the late gene region of the phage CBA120 genome.

(2) The recombinant phage according to paragraph 1, wherein the recombinant phage specifically infects Escherichia coli O157:H7.

(3) The recombinant phage of paragraph 1 or 2, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates a signal upon reaction with a substrate.

(4) The recombinant phage of any one of paragraphs 1-3, which further comprises a codon-optimized untranslated region upstream of the indicator gene, wherein the untranslated region includes a phage late gene promoter and a ribosome entry site.

(5) A method for preparing a recombinant indicator phage, comprising: selecting a wild-type phage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid/vector comprising an indicator gene; transforming the homologous recombination plasmid/vector into a target pathogenic bacterium; The selected wild-type phage infects the transformed target pathogen, allowing homologous recombination between the plasmid/vector and the phage genome; and isolation of specific clones of the recombinant phage.

(6) The method of paragraph 5, wherein preparing the homologous recombination plasmid/vector comprises: determining the native nucleotide sequence in the late genome region of the selected phage; annotating the genome and identifying the major capsid protein gene of the selected phage; sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene; and incorporation of sequences designed for homologous recombination into the plasmid/vector.

(7) The method of paragraph 5 or 6, wherein designing the sequence further includes inserting an untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site upstream of the codon-optimized indicator gene.

(8) The method of any of paragraphs 5-7, wherein the homologous recombination plasmid comprises an untranslated region upstream of a codon-optimized indicator gene comprising a phage late gene promoter and a ribosome entry site.

(9) The method of any one of paragraphs 5-8, wherein the wild-type phage is CBA120, and the target pathogen is Escherichia coli O157:H7.

(10) The method of any of paragraphs 5-9, wherein isolating specific clones of the recombinant phage comprises a limiting dilution assay for isolating clones demonstrating expression of the indicated gene.

(11) A method for detecting Escherichia coli O157:H7 in a sample, comprising: incubating the sample with a recombinant phage derived from CBA120, and detecting an indicator protein product produced by the recombinant phage, wherein positive detection of the indicator protein product indicates the presence of Escherichia coli O157:H7.

(12) The method of paragraph 11, wherein the sample is a food, environmental, water, commercial or clinical sample.

(13) The method of paragraph 11 or 12, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in a sample of a standard size used in the food safety industry .

(14) The method of any of paragraphs 11-13, wherein the sample includes beef or vegetables.

(15) The method of any of paragraphs 11-14, wherein the sample is first incubated under conditions that favor enriched growth for 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.

(16) The method of any of paragraphs 11-15, wherein the total time to obtain the result is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours or less than 6 hours.

(17) The method of any of paragraphs 11-16, wherein the ratio of signal to background produced by the detection of the indicator is at least 2.0 or at least 2.5.

(18) A kit for detecting Escherichia coli O157:H7 comprising a recombinant phage derived from CBA120.

(19) The kit of paragraph 18, further comprising a substrate for reacting with the indicator to detect the soluble protein product expressed by the recombinant phage.

(20) A system for detecting Escherichia coli O157:H7 comprising a recombinant phage derived from CBA120.

Example

The results described in the examples below demonstrate the detection of small numbers of cells, even single bacteria, within a shortened time to result.

Example 1. Production of indicator phages from CBA120

The indicator phage CBA120NanoLuc was generated by homologous recombination using the following detailed procedure, as shown in Figures 1-3.

The genome sequence of the CBA120 phage is available on GenBank at the National Center for Biotechnology Information, submitted under "Escherichia phage Cba120" ID 12291. The genome was fully annotated, although most genes were marked as 'hypothetical proteins', indicating automatic open reading frame discovery. It is assumed that the protein only needs to have start and stop codons and may not be expressed because DNA regulation (promoter/enhancer/operon etc.) is not defined in the sequence.

Late gene regions identified by comparison with other phage genomes. CBA120 and all other ViI-like phages belong to the ViI-like phage group (ViIviruses or ViIviruses), which are related to T4-like phages. Phage T4 is the most studied phage and many gene homologues can be found and are tagged as such. This includes the late gene region, which consists of highly expressed phage structural proteins. This region is targeted for insertion reporter gene. The main capsid protein was specifically identified. Since the major capsid protein usually has the highest expression, inserting the reporter directly downstream of the major capsid protein can maximize reporter expression.

Design the sequence to insert a codon-optimized Gene. As shown in Figure 2, a homologous recombination (HR) plasmid was designed initially with 500 bp upstream and downstream of the insertion point. Previous HR plasmids using firefly luciferase as a reporter gene produced poor transformation, which was mitigated by use of a shorter downstream region. Presumably, the entire 500bp region selected has a toxic effect in bacteria. Therefore, the modified downstream region only extends about 300 bp.

The upstream region consists of the 3' end of the major capsid protein, where the insertion follows the stop codon (TAA): SEQ ID NO: 1

Ctttcatgctggaagttgaagcgaacggtatcggtgttgacacccgtcgtggtaaaggcaaccgtgttctgtgttctccgaacgtggcatccgctctggcgatgtctggcatgctggactatgctccggttctgcaggaaaacactaaactggctgttgacccgactggccagaccttcgctggtgttctgtccaacggtatgcgcgtctatgttgacccgtatgctgtagcagaatatatcaccctggcatacaaaggcgcaactgcgctggatgccggtatcttcttcgcgccgtatgtgccgctggaaatgtaccgcacccagggtgaaaccaccttcgctccgcgtatggcgttcaaaacccgttacggcatctgtgctaacccgttcgtacagattccggctaaccaagacccgcaggtttacgtgactgctgacggtattgctcaagacagcaacccgtatttccgcaaaggtctgatcaaatctctgttctaa

This was followed by a MluI restriction site, followed by the T4 late gene promoter consensus sequence consisting of the ^-10σ70 factor consensus binding sequence (CTAAATAcCcc (SEQ ID NO: 2)). The promoter was designed based on the synthetic known -10 sequence. This was followed by insertion of 14 random base pairs, a ribosome entry site, the Shine-Dalgarno consensus sequence (aaggaggt), followed by 6 random base pairs. Random base pairs were chosen to maintain similar GC content to other upstream untranslated regions. SEQ ID NO: 3

acgcgtCTAAATAcCccaaatactagtagataaggaggttttcga

Inserted a codon-optimized version of Promega from pNL1.3 with the secretion signal SEQ ID NO: 4

ATGAATAGCTTTAGCACCAGCGCCTTTGGCCCTGTTGCCTTTAGCCTGGGCCTGCTGCTGGTTCTGCCGGCAGCATTTCCGGCCCCGGTGTTCACCCTGGAAGATTTTGTGGGCGATTGGCGCCAGACCGCCGGTTATAACCTGGATCAGGTGCTGGAACAGGGTGGTGTGAGCAGCCTGTTTCAGAATCTGGGCGTGAGCGTGACCCCGATTCAGCGCATTGTGCTGAGCGGCGAGAACGGCCTGAAAATTGATATTCATGTTATTATTCCGTATGAGGGTCTGAGCGGCGATCAGATGGGCCAGATTGAAAAAATCTTTAAGGTGGTGTATCCGGTGGACGACCATCATTTCAAGGTGATCCTGCATTACGGCACACTGGTGATTGACGGCGTTACCCCGAACATGATCGACTATTTCGGCCGCCCGTATGAAGGTATCGCCGTGTTCGACGGCAAGAAAATTACCGTGACCGGTACCCTGTGGAACGGCAACAAGATCATTGACGAGCGCCTGATTAACCCGGATGGTAGCCTGCTGTTTCGCGTGACCATTAATGGCGTGACCGGCTGGCGTCTGTGTGAACGCATCCTGGCCTAA

Next is the 298bp downstream HR fragment, which contains the putative gene. SEQ ID NO: 5

gcgacaggttttgataacaaaccccgcttcggcggggtttttctttatagggatatgtaagataataaagcctcatttatcaaaggaggttaaaatgtctcatcaattatctggcggtgcagtcgatactctattcgttcttttctggtttggacctcgtgaagctggggaaatacctgctaaatctggagaagccgaattggcctccctggggttttgtaaacgagttgatgttaaaaacgtaccaaaaggtcgagatacacatctgtgtgtactcaccgaggaaggttacaaatac

After this, a consensus transcription terminator is inserted together with a stop codon, which should only function on the plasmid to reduce any readthrough and possible toxic effects. Since homologous recombination occurs only in the HR region, transcription terminators should not be included in the recombinant phage. SEQ ID NO: 6taaTTTGATAACAAACCCCGCTTCGGCGGGGTTTTTCTTTTATAGG

The complete sequence was synthesized as a plasmid (GeneWiz). Transform the plasmid into pre-prepared E. coli O157:H7 electroporation-competent cells using the protocol contained in the Bio-Rad MicroPulser Electroporation Apparatus Instructions and Application Note (Cat. No. 165-2100).

The synthesized plasmid DNA (pUC57.CBA.HR.NanoLuc) (4 μg plasmid DNA) was dissolved in autoclaved filtered deionized water (40 μL) to prepare a 100 ng/μL stock solution. This plasmid (1 μL) was mixed with 20 μL of thawed (on ice) E. coli O157:H7 electroporation competent cells (derived from avirulent E. coli O157:H7 bacteria, ATCC 43888). The cell+DNA mixture was transferred to an ice-cold Bio-Rad 0.1 cm electroporation cuvette and subjected to the MicroPulser Electroporation Apparatus using program Ecl. The mixture was immediately transferred to 1 mL recovery medium (Life Technologies) and incubated at 42°C, 220 rpm for 1 hour.

Aliquots of 1 μL, 100 μL and the remaining culture concentrated by centrifugation (2 min, 6800 g) and resuspended in 100 μL were inoculated onto selection medium (LB+Amp agar plates from Teknova) and incubated overnight at 37 °C .

The next day, 23 colonies (+1 negative control) were screened by inoculating 100 μL LB+Amp and incubating at 37° C. for 2.5 hours, followed by luciferase activity. Dispense 5 μL of each culture to Promega Luciferase assay, and the Promega Read on a 96 fluorometer. All 23 colonies were positive.

The top 3 wells were pooled and seeded into 4 mL LB+Amp and grown to ^1.8x107 cells/mL. Bacteria were infected with wild-type CBA120 phage from the Kutter lab (see Kutter et al., Virology Journal 2011, 8:430) at an MOI of 0.1, and the homologous recombination infection was incubated at 37°C for 3 hours.

Bacteria concentrations were monitored for 4 hours; bacteria doubled at 2 hours and then began to decline, indicating successful phage infection.

Example 2. Separation of CBA120NanoLuc

Following homologous recombination to generate recombinant phage genomes, a series of titer and enrichment steps were used to isolate expressed specific recombinant phage.

To reduce background from plasmid expression To signal, the lysate was washed 3 times with TMS in an Amicon UltraConcentrator, spun to concentrate the volume from 4 mL to 500 μL; TMS was added to bring the volume to 4 mL, and the series was repeated.

To determine the initial ratio of recombinant to wild-type phage, a limiting dilution assay based on TCID50 (tissue culture infectious dose 50%) was used to determine the concentration of infectious units (IU/mL), similar to the number of viral particles or plaque-forming units, and Determine the number of luciferase transducing units (TU/mL). In these assays, samples were serially diluted and each dilution was aliquoted into replicate wells with E. coli O157:H7 bacteria. Any well showing luciferase activity must have been infected with at least one recombinant phage. Any well showing cell lysis has been infected with at least one phage. Based on the highest dilution that occurred in each case, the original concentration was recalculated. These initial phage mixtures from transformed cells typically yielded a ratio of 20,000 wild-type IU for each recombinant phage TU. Steps are then taken to isolate and amplify the recombinant phage.

As shown in Figure 4, in some experiments recombinant phage were isolated from a mixture containing 0.83% of total phage. The phage mixture was diluted into 96-well plates to obtain an average of 3 recombinant TUs per plate, which corresponds to approximately 3.8 infectious units (IU) of predominantly wild-type phage per well. Bacteria were added such that each well contained 50 μL of turbid E. coli O157:H7. After incubation for 2 hours at 37°C, wells were sampled and screened for the presence of luciferase. Any positive wells may have been inoculated with a single recombinant phage, and at this stage the mixture contained an enrichment ratio of 1 recombinant phage:3.8 wild-type phage, which is an enrichment higher than the original 1:120 ratio. Of the 96 wells screened, 7 were positive. No further rounds of limiting dilution assays were required in this experiment.

A plaque assay was performed in which plaques were individually picked and screened for luciferase transduction ability, ensuring that there were approximately 3 recombinants in the screened plaque mixture. Suspend each plaque in 100 μL TMS and add 5 μL to wells containing turbid E. coli O157:H7 cultures and assay wells after incubation at 37 °C for 45 min to 1 h.

Positive wells were expected to contain pure cultures of recombinant phage, but underwent another round of plaque purification. Finally, large-scale production is performed to obtain high-titer stocks suitable for E. coli O157:H7 detection assays. Phage particles were separated from contaminating luciferase proteins using cesium chloride isopycnic gradient centrifugation to reduce background.

Example 3. Bacterial detection using CBA120NanoLuc indicator phage

Detection of E. coli O157:H7 using the CBA120NanoLuc indicator phage was tested in an experiment using the basic assay format shown in FIG. 6 . First, 1-10,000 cells were removed from the culture and infected with 10 ⁵ , 10 ⁶ and 10 ⁷ phage/mL in LB of the same sample volume for 2 hours. Add lysis buffer and After reagents, use 96 instrument read responses. Figure ⁷ shows that the highest signal/background ratio was achieved with 106 phage/mL used to infect samples.

Figure 8 shows data from 6-10 replicates, each using the same number of cells from cell cultures in LB. Phage at a concentration of ¹⁰⁶ phage/mL was used to infect the samples, and the infected cells were incubated at 37°C for 2 hours. Add lysis buffer and After reagents, use 96 instrument read responses. Figure 8 shows that CBA120NanoLuc can detect a single (1) cell with a signal significantly above background.

Figure 9 shows based on the data in Figure 8 that CBA120NanoLuc can detect a single (1) E. coli O157:H7 cell with a signal-to-background ratio >2.0.

The performance of CBA120NanoLuc indicator phage for detection of Escherichia coli O157:H7 was also certified by AOAC Institute (Certificate No. 081601) on August 1, 2016.

Example 4. Bacterial Detection Using CBA120NanoLuc in Beef Assays

CBA120NanoLuc for the detection of E. coli O157:H7 in beef assays. For all beef experiments, 50 RLU was used as background value, and 3 times background value was considered positive (ie, >150 RLU is positive, or signal/background >3.0). Compared to the second confirmation method described below, there were no false positives or negatives.

For a 25 g beef sample, pre-warmed TSB medium (42 °C) was added to the sample to a 1:3 sample:medium (25 g:75 mL). Samples were mixed with a Stomacher on low setting and/or equivalent for 30 s, then incubated at 42°C without shaking. Close the bag by folding over the top 2-3 times and clipping closed. After enrichment at 42°C for 5 hours (for 10 mL aliquots in the next step) or 6 hours (for 1 mL aliquots in the next step), gently massage the bag to mix the contents thoroughly.

Aliquots of 1 mL or 10 mL were removed from the bags for testing. These correspond to "1 mL concentration" or "10 mL concentration" in the data for all beef assay experiments in Figures 10-16.

Centrifuge a 10 mL aliquot at 3400 g for 5 min, discard the supernatant, and resuspend the contents in 1 mL of pre-warmed TSB. Add CBA120 indicator phage to infect any target bacteria in the sample by adding 10 µL of 1x ¹⁰ phage/mL.

Centrifuge a 1 mL aliquot in a microcentrifuge at top speed for 1 min, discard the supernatant, and resuspend the contents in 200 µL of pre-warmed TSB. To infect target bacteria, add ¹⁵ µL of 1.2x 107 phage/mL of CBA120 indicator phage.

Samples with CBA120 indicator phage were incubated at 37°C for 2 hours, vortexed briefly, centrifuged for 5-10 seconds to pellet debris, and 150 μL of sample was transferred to a 96-well plate (be careful not to disturb the debris pellet). Add lysis buffer (10 μL) to each well and mix gently by pipetting. freshly prepared Reagent (50 μL) was added to each well and mixed gently by pipetting (or auto-injection). (Will Luciferase assay substrate diluted 1:50 to Prepare luciferase assay buffer Reagents, e.g., to prepare 1 mL Reagent, 20 μL Add 1 mL of luciferase assay substrate luciferase assay buffer)

Plates were read on a GLOMAX 96 instrument 3 minutes after substrate addition.

Second confirmation method:

Use the antibody coated with O157 ( Life Technologies #71004) of the particles of immunomagnetic separation (IMS) and in selective plate ( plates, BD #214984) for confirmation of E. coli O157:H7 on overnight enriched cultures.

For readiness confirmation, samples were incubated overnight at 42°C ± 1° (for a total of 18-24 hours or an additional 13-19 hours). Remove 1 mL from the overnight culture and proceed to Anti-E. coli O157 procedure. Briefly, 20 μL of IMS particles were added to diluted overnight cultures and incubated for 10 min at room temperature. The magnetic particles were separated by a magnet for 3 minutes, and then washed 3 times with 1 ml of PBS. After the last wash, apply the granules to the plate (BD#214984) and incubated at 37°C±1° for 18-24 hours.

Rufous-brown colonies (presumptive positives) were grown overnight (18-24 hours) in TSB medium at 37°C ± 1° for serological confirmation. The presence of O157 and H7 antigens was determined using an agglutination assay (Remel Wellcolex E. coli O157:H7#R30959601). Follow the manufacturer's instructions, using 40 μL of overnight culture. The results confirm the presence or absence of O157 and/or H7 antigens and provide confirmation for E. coli O157:H7.

Data from a 25 g beef sample are shown in Figures 10-12. Figures 10-11 correspond to 1 mL concentration of the enriched sample, and Figure 12 corresponds to 10 mL concentration of the enriched sample. All positives were detected after 6 hours enrichment at 1 mL concentration and after 5 hours enrichment at 10 mL concentration. Figure 11-12 shows the pass / Confirmation of planking.

For larger (125 g) beef samples, experiments were performed with ground beef and shredded beef. The procedure is similar except that ground beef samples need to be processed with the stomacher for at least 120 seconds on high setting and/or equivalent. Enrichment of both samples was then carried out at 42°C for 8 hours, and the rest of the steps were as described above.

Data from a 125 g beef sample are shown in Figures 13-16. Figures 13 and 15 correspond to the 1 mL concentration and Figures 14 and 16 correspond to the 10 mL concentration. Figure 15-16 shows the pass / Confirmation of planking. All positives were detected after 7 hours of enrichment.

Example 5. Vegetable washing assay

Data from the spinach wash filter assay is shown in Figure 17, which shows that the assay can detect 1 E. coli O157:H7 cell in 100 mL of spinach wash after 3 hours of enrichment. According to the manufacturer's instructions, by using on the overnight culture of each sample / Tests were used to confirm these results, as described above under "Second Confirmation Method".

To prepare vegetable washes, weigh vegetable leaves (such as spinach or lettuce) and add them to clean plastic bags. Add 5 mL of water per gram (g) of vegetables. Mix the leaves and solution by hand for a few minutes. The liquid is then extracted from the plastic bag and used as "vegetable wash". Using this method, approximately 1 million bacteria were found by CFU to be present on a single spinach leaf (1-2 g).

Next, vacuum filter 100 mL of "vegetable wash" through a 47 mm 0.45 μΜ filter. Remove the filter and place it in a small resealable plastic bag. Pre-warmed (42°C) TSB medium (600 μL) was added to the bag to cover the filter. The filters were then incubated at 42 °C for 3 h with gentle agitation. An aliquot (300 μL) of the enrichment medium was removed for confirmation purposes. The CBA120NanoLuc indicator phage was then added to the remaining medium in the bag to a final concentration of ¹ x 106 phage/mL, the bag was gently agitated, and then incubated at 37°C for 2 hours. Finally, transfer 100-150 μL of the infection reaction to a 96-well plate. Add lysis buffer (10 μL) and prepared Reagent (50 μL), and in the luminometer ( 96) read the sample.

Figure 17 shows data from spinach washing assays, including / Confirmation results for planking. The ability to discriminate a single target bacterial cell from 105 non ^- target bacteria in plant washes was surprising and again demonstrated the specificity and sensitivity of the assay.

sequence listing

<110> American Holdings Laboratory Corporation

Gil, Jose S.

Erickson, Stephen

Hopkins, Ben Barrett

Nguyen, Minh Mindy Bao

Anderson, Dwight Lyman

<120> Method and system for rapid detection of microorganisms using infectious agents

<130> 1035526

<150> US 62/280,043

<151> 2016-01-18

<150> US 62/280,465

<151> 2016-01-19

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 500

<212>DNA

<213> Viruses of the genus ViI

<400> 1

ctttcatgct ggaagttgaa gcgaacggta tcggtgttga cacccgtcgt ggtaaaggca 60

accgtgttct gtgttctccg aacgtggcat ccgctctggc gatgtctggc atgctggact 120

atgctccggt tctgcaggaa aacactaaac tggctgttga cccgactggc cagaccttcg 180

ctggtgttct gtccaacggt atgcgcgtct atgttgaccc gtatgctgta gcagaatata 240

tcaccctggc atacaaaggc gcaactgcgc tggatgccgg tatcttcttc gcgccgtatg 300

tgccgctgga aatgtaccgc accccagggtg aaaccacctt cgctccgcgt atggcgttca 360

aaacccgtta cggcatctgt gctaacccgt tcgtacagat tccggctaac caagacccgc 420

aggtttacgt gactgctgac ggtattgctc aagacagcaa cccgtatttc cgcaaaggtc 480

tgatcaaatc tctgttctaa 500

<210> 2

<211> 11

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic Consensus Binding Sequence

<400> 2

ctaaataccc c 11

<210> 3

<211> 45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic phage nucleotide sequence

<400> 3

acgcgtctaa ataccccaaa tactagtaga taaggaggtt ttcga 45

<210> 4

<211> 600

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic phage nucleotide sequence

<400> 4

atgaatagct ttagcaccag cgcctttggc cctgttgcct ttagcctggg cctgctgctg 60

gttctgccgg cagcatttcc ggccccggtg ttcaccctgg aagattttgt gggcgattgg 120

cgccagaccg ccggttataa cctggatcag gtgctggaac agggtggtgt gagcagcctg 180

tttcagaatc tgggcgtgag cgtgaccccg attcagcgca ttgtgctgag cggcgagaac 240

ggcctgaaaa ttgatattca tgttattatt ccgtatgagg gtctgagcgg cgatcagatg 300

ggccagattg aaaaaatctt taaggtggtg tatccggtgg acgaccatca tttcaaggtg 360

atcctgcatt acggcacact ggtgattgac ggcgttaccc cgaacatgat cgactatttc 420

ggccgcccgt atgaaggtat cgccgtgttc gacggcaaga aaattaccgt gaccggtacc 480

ctgtggaacg gcaacaagat cattgacgag cgcctgatta acccggatgg tagcctgctg 540

tttcgcgtga ccattaatgg cgtgaccggc tggcgtctgt gtgaacgcat cctggcctaa 600

<210> 5

<211> 298

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic phage nucleotide sequence

<400> 5

gcgacaggtt ttgataacaa accccgcttc ggcggggttt ttctttatag ggatatgtaa 60

gataataaag cctcatttat caaaggaggt taaaatgtct catcaattat ctggcggtgc 120

agtcgatact ctattcgttc ttttctggtt tggacctcgt gaagctgggg aaatacctgc 180

taaatctgga gaagccgaat tggcctccct ggggttttgt aaacgagttg atgttaaaaa 240

cgtaccaaaa ggtcgagata cacatctgtg tgtactcacc gaggaaggtt acaaatac 298

<210> 6

<211> 45

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic phage nucleotide sequence

<400> 6

taatttgata acaaaccccg cttcggcggg gttttcttt atagg 45

Claims (20)

1. A recombinant phage comprising an indicator gene inserted into the late gene region of the phage CBA120 genome. 2. The recombinant phage according to claim 1, wherein said recombinant phage specifically infects Escherichia coli O157:H7. 3. The recombinant phage of claim 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates a signal upon reaction with a substrate. 4. The recombinant phage of claim 3, further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a phage late gene promoter and a ribosome entry site. 5. A method for preparing recombinant indicator phage, comprising: Select wild-type phages that specifically infect target pathogenic bacteria; Preparation of homologous recombination plasmids/vectors containing indicator genes; Transform homologous recombination plasmids/vectors into target pathogenic bacteria; Infect the transformed target pathogen with the selected wild-type phage, allowing homologous recombination between the plasmid/vector and the phage genome; and Isolation of specific clones of recombinant phage. 6. The method of claim 5, wherein preparing a homologous recombination plasmid/vector comprises: Determining the native nucleotide sequence in the late region of the genome of the selected phage; Annotate genomes and identify major capsid protein genes of selected phages; A sequence designed for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene; and Sequences designed for homologous recombination are incorporated into the plasmid/vector. 7. The method of claim 6, wherein designing the sequence further comprises inserting an untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site upstream of the codon-optimized indicator gene. 8. The method of claim 5, wherein the homologous recombination plasmid comprises an untranslated region upstream of a codon-optimized indicator gene comprising a phage late gene promoter and a ribosome entry site. 9. The method of claim 5, wherein the wild-type phage is CBA120, and the target pathogen is Escherichia coli O157:H7. 10. The method of claim 5, wherein isolating specific clones of the recombinant phage comprises a limiting dilution assay for isolating clones demonstrating expression of the indicated gene. 11. A method for detecting Escherichia coli O157:H7 in a sample, comprising: incubating the sample with recombinant phage derived from CBA120, and Detect the indicator protein product produced by the recombinant phage, wherein the positive detection of the indicator protein product indicates the presence of E. coli O157:H7 in the sample. 12. The method of claim 11, wherein said sample is a food, environmental, water, commercial or clinical sample. 13. The method of claim 11, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacteria in a standard size sample used in the food safety industry. 14. The method of claim 12, wherein said sample comprises beef or vegetables. 15. The method of claim 11, wherein said sample is first incubated under conditions conducive to enriched growth for a period of 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. 16. The method of claim 11, wherein the total time to result is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours or less than 6 hours. 17. The method of claim 11, wherein the ratio of signal to background produced by detection of the indicator is at least 2.0 or at least 2.5. 18. A kit for detecting Escherichia coli O157:H7 comprising a recombinant phage derived from CBA120. 19. The kit of claim 18, further comprising a substrate for reacting with an indicator to detect a soluble protein product expressed by said recombinant phage. 20. A system for detecting E. coli O157:H7 comprising a recombinant phage derived from CBA120.