Self-contained device and system for the detection of microorganisms - Patents.com

Abstract

Disclosed herein are devices, methods, and systems for rapid detection of microorganisms using recombinant bacteriophages. The specificity of the recombinant bacteriophage's binding to microorganisms allows for highly specific detection of targeted microorganisms of interest. The devices may contain separate compartments, which may be configured to allow a user to combine contents from different compartments at various stages of the detection method. For example, a sample to be tested may be combined with bacteriophage in one compartment, and the contents may be incubated for some period of time before being added to the contents (such as a substrate) of another compartment of the device.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 777,473, filed December 10, 2018, and 62 / 798,980, filed January 30, 2019. The disclosures of U.S. Patent Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, 15 / 409,258, 16 / 298,695, and U.S. Provisional Application Nos. 62 / 616,956, 62 / 628,616, 62 / 661,739, 62 / 640,793, and 62 / 798,980 are incorporated by reference in their entireties herein.

[0002] FIELD OF THEINVENTION The present invention relates to methods, apparatus, devices and systems for detecting microorganisms of interest using recombinant bacteriophages. [Background technology]

[0003] background There is a great 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 affect many humans and livestock and can also result in significant economic losses. Given the dramatic increase in life-threatening or fatal illnesses resulting from the ingestion of food contaminated with certain microorganisms (e.g., Staphylococcus spp., Escherichia coli, or Salmonella spp.), microbial detection is a high priority for the Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC).

[0004] Traditional microbiological tests for bacterial detection rely on non-selective and selective enrichment culture followed by plating on selective media and further testing to confirm suspected colonies. Such procedures can take several days. Various rapid methods have been investigated and introduced to reduce the turnaround time. However, previous turnaround time reduction methods have drawbacks. For example, techniques involving direct immunoassays and gene probes generally require an overnight enrichment step to obtain adequate sensitivity and therefore do not provide results on the same day. Also, polymerase chain reaction (PCR) tests involve an amplification step and therefore can achieve both very high sensitivity and selectivity; however, the sample size that can be economically subjected to PCR testing is limited. Dilute bacterial suspensions that can be subjected to PCR likely do not contain cells, and therefore purification and / or time-consuming enrichment steps are still required.

[0005] The time required for traditional biological enrichment is dictated by the growth rate of the target bacterial population of the sample, the effect of the sample matrix, and the required sensitivity. In fact, the most sensitive methods use overnight incubation, taking about 24 hours overall. Due to the time required for culture, these methods can take up to three days depending on the organism to be identified and the source of the sample. This time delay is generally unsuitable because such a delay could contaminate food or water or other products that are introduced into livestock or humans. Furthermore, the rise of antibiotic resistance and biodefense concerns have made the rapid identification of bacterial pathogens in water, food, and clinical samples a significant priority worldwide.

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

[0007] Abstract Embodiments of the present invention include devices, compositions, methods, apparatus, systems, and kits for the detection of microorganisms.The present invention can be embodied in a variety of ways.

[0008] Aspects of the present invention include devices for facilitating simple and straightforward microbial detection. Described herein is an apparatus or device for performing an assay for detecting a microorganism in a test sample using recombinant bacteriophage.

[0009] In one aspect, the present invention utilizes the high specificity of bacteriophage that can bind to microorganisms and detect low levels of microorganisms. In some embodiments, the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or single bacteria in a sample, based on food safety industry standard sizes. In other embodiments, the sample is first incubated in favorable growth conditions for an enrichment 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. In some embodiments, the sample is not enriched before incubation with recombinant bacteriophage.

[0010] In some embodiments, the recombinant bacteriophage is genetically modified to contain a reporter gene as previously described, hi further embodiments, the recombinant bacteriophage is derived from a bacteriophage specific for the microorganism to be detected. For example, the recombinant bacteriophage can be derived from any of the following bacteriophages: Salmonella phage SPN1S, Salmonella phage 10, Salmonella phage ε15, Salmonella phage SEA1, Salmonella phage Spn1s, Salmonella phage P22, Listeria phage LipZ5, Listeria phage P40, Listeria phage vB_LmoM_AG20, Listeria phage P70, Listeria phage A511, Staphylococcus phage P4W, Staphylococcus phage K, Staphylococcus phage Twort, Staphylococcus phage SA97, or E. coli O157:H7 phage CBA120.

[0011] In some embodiments, the recombinant bacteriophage may be stabilized or preserved, for example, the recombinant bacteriophage may be lyophilized.

[0012] In certain embodiments, the reporter generates an indicator moiety. In certain embodiments, the indicator moiety can generate an intrinsic signal. In other embodiments, the indicator moiety includes an enzyme that generates a signal upon reaction with a substrate. In yet other embodiments, the indicator moiety includes a cofactor that generates a signal upon reaction with one or more additional signal-generating components. For example, the indicator moiety includes at least one of a fluorophore, a fluorescent protein, a particle, and an enzyme. The enzyme can include at least one of a luciferase, a phosphatase, a peroxidase, and a glycosidase. The luciferase gene can be a naturally occurring gene (such as Oplophorus luciferase, firefly luciferase, Lucia luciferase, or Renilla luciferase) or can be a genetically engineered gene.

[0013] Some embodiments of the invention include devices or apparatus for carrying out detection methods. In some embodiments, the device may include separate compartments. In some embodiments, the separate compartments of the device may be configured to allow the user to combine contents from different compartments at various stages of the detection method. For example, the sample to be tested may be combined with bacteriophage in one compartment and incubated for some period of time before adding the contents (such as a substrate) to the contents of another compartment of the device. In such embodiments, the substrate may react with any reporter (or indicator moiety) produced as a result of infection with the recombinant bacteriophage (e.g., if the target microorganism is present in the sample).

[0014] Some embodiments include a device comprising a first compartment containing a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, said construct comprising a promoter and an indicator gene; and a second compartment containing a signal detection component, said signal detection component facilitating detection of said indicator gene product produced as a result of infection of said sample with said recombinant bacteriophage. In some embodiments, the signal detection component is a substrate and the indicator gene encodes an enzyme. In some embodiments, the enzyme is luciferase.

[0015] In some embodiments, the device includes a first compartment that includes a recombinant bacteriophage, an inlet / portal for adding a portion of the test sample to the recombinant bacteriophage, and a second compartment that includes a substrate or other paired reagent, and the method further includes adding the substrate from the second compartment to the sample simultaneously with or after adding the recombinant bacteriophage. In some embodiments, the device includes a third compartment. The third compartment can include, for example, a growth medium to enrich the sample. In other embodiments, the device does not include a medium.

[0016] In some embodiments, the device further comprises a second compartment comprising a substrate, wherein detecting the indicator gene product is by contacting the indicator gene product with the substrate. In some embodiments, the solid support is a bead. In some embodiments, the solid support comprises polyethylene (PE), polypropylene (PP), polystyrene (PS), polylactic acid (PLA), and polyvinyl chloride (PVC). In some embodiments, the solid support is dried prior to contact with the sample. In some embodiments, the solid support is immersed in a medium prior to contact with the sample. In some embodiments, the solid support capturing one or more organisms is incubated with a growth medium in the third compartment prior to contact with the recombinant bacteriophage.

[0017] In some embodiments, the solid support is coated with a cell-binding moiety that binds with high affinity to the microorganism of interest in the sample, allowing more bacteria to be bound to the solid support, increasing the sensitivity and specificity of the assay. In other embodiments, the solid support is coated with an antibody that can bind to the microorganism of interest.

[0018] In some embodiments, the first compartment comprises a seal, where contact of the recombinant bacteriophage with the sample disrupts the seal, such that upon disruption of the seal, the recombinant bacteriophage from the first compartment contacts the sample and infects one or more microorganisms in the sample, thereby producing an indicator gene product (indicator protein). In further embodiments, the device comprises a stop-lock for stepwise mixing of the medium, recombinant bacteriophage, and substrate with the sample.

[0019] In some embodiments, the indicator gene product comprises at least one of a fluorophore, a fluorescent protein, a particle, and an enzyme. In some embodiments, the enzyme comprises at least one of a luciferase, a phosphatase, a peroxidase, and a glycosidase. In some embodiments, the luciferase is an engineered luciferase. In some embodiments, the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0020] In another embodiment, a method for detecting one or more microorganisms of interest in a sample comprises contacting the sample with a solid support of an apparatus, the solid support capturing, if present, the one or more microorganisms in the sample, wherein the apparatus comprises a first compartment comprising a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, the construct comprising a promoter and an indicator gene, contacting the recombinant bacteriophage from the first compartment with the sample such that the recombinant bacteriophage infects one or more microorganisms in the sample, thereby producing an indicator gene product, and detecting the indicator gene product.

[0021] In some embodiments, the method includes incubating the solid support capturing one or more microorganisms of interest in a growth medium for a period of time prior to adding the recombinant bacteriophage. In some embodiments, the incubation is for 0-2 hours. In some embodiments, the bacteriophage is contacted with the sample for 0.5-3 hours prior to detection of the indicator gene product.

[0022] In some embodiments, the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a sample relative to food safety industry standard sizes. In some embodiments, the sample comprises meat or vegetables. In some embodiments, the sample is a food sample, a water sample, a dairy sample, an environmental sample, a commercial sample, or a clinical sample.

[0023] In some embodiments, samples are first incubated in preferred growth conditions for an enrichment 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.

[0024] Further embodiments include systems and kits for detection of microorganisms (such as Listeria, Salmonella, Staphylococcus, or E. coli O157:H7) that include the recombinant bacteriophage. Some embodiments further include a substrate for reaction with an indicator moiety of the recombinant bacteriophage. These systems or kits can include features described for the bacteriophages, compositions, and methods of the invention. In yet other embodiments, the invention includes non-transitory computer readable media for use with the methods or systems according to the invention.

[0025] In another aspect, the disclosure provides a system for detecting a microorganism of interest in a sample, the system comprising: a device; a first compartment comprising a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, the construct comprising a promoter and an indicator gene; and a signal detection component, the signal detection component capable of detecting an indicator gene product produced from infection of the recombinant bacteriophage into the sample. In some embodiments, the signal detection component is a handheld luminometer.

[0026] The invention can be better understood with reference to the following non-limiting drawings. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is one embodiment of a method for detecting a bacterium of interest using a recombinant bacteriophage.

[0028] [Diagram 2] Figure 2 shows the results of detection of L. monocytogenes cultures using one embodiment of a self-contained device with a swab as the solid support. Signals corresponding to the presence of bacteria were detected by a Hygiena luminometer, a GloMax luminometer, and a GloMax20 / 20 luminometer. Table 1 shows the results of log phase cultures and Table 2 shows the results of overnight cultures.

[0029] [Diagram 3] Figures 3A and 3B are plots generated from the data shown in Table 1. Figure 3A shows measurements of the signal detected using the Hygiena. Swabs were inoculated with log phase cells at the indicated CFU levels. Samples were immediately infected with Listeria phage cocktail for 4 hours. Substrate was added and samples were read in the Hygiena luminometer. A signal above 10 RLU was considered positive. Using this method, approximately 25,000 CFU are required to obtain a positive result.

[0030] FIG. 3B shows measurements of signal detected using a GloMax20 / 20 luminometer and a GloMax (also known as GloMax96) luminometer. Swabs were inoculated with log-phase cells at the indicated CFU levels. Samples were immediately infected with Listeria phage cocktail for 4 hours. Substrate was added and samples were read on either a GloMax20 / 20 luminometer (1 mL sample) or a GloMax (150 μl sample) luminometer. A signal / background ratio of >3.0 is considered positive. Using this method, approximately 5,000 CFU are required to obtain a positive result.

[0031] [Figure 4] Figure 4 shows the detection of Salmonella in ground turkey inoculated with Salmonella. Table 3 shows the uninoculated control samples and Table 4 shows the uninoculated turkey samples. The study was repeated using various inoculations and infection times.

[0032] [Diagram 5] Figures 5A and 5B are plots made from the data in Figure 4. Figure 6A shows that turkey samples inoculated with Salmonella were detected as positive at every incubation and infection time tested. Turkey samples were grown at 41°C for 24 hours after inoculation and then tested using the methods disclosed in this application. For relative signal: 0 hour incubation, 2 hour infection > 1 hour incubation, 0.5 hour infection > 0 hour incubation, 0.5 hour infection. Additionally, a comparison of the RLU signals shows that the GloMax luminometer shows a significantly higher signal than the Hygiena luminometer.

[0033] Figure 5B shows that detection using the GloMax20 / 20 luminometer and the GloMax luminometer showed similar signal / background ratios for the same samples. The GloMax20 / 20 showed a larger signal (Figure 5A), but the background was significantly higher than the GloMax. Thus, when determining signal / background, the two luminometers perform similarly.

[0034] [Figure 6] Figure 6 shows data for detection of Salmonella in three turkey samples (samples 21, 24, and 26) that were inoculated with Salmonella prior to assay using the self-contained device. Samples were infected for different times as indicated prior to detection of signal.

[0035] [Figure 7]Figures 7A-7C are plots constructed from the data presented in Figure 6. Figures 7A-7C show the results of an experiment in which three inoculated ground turkey samples were enriched for 24 hours, swabbed, and assayed. Samples 24 (Figure 7B) and 26 (Figure 7C) showed no signal by the Hygiena handheld luminometer for the sample infected with phage for 30 minutes, but did show a signal for the sample infected for 2 hours. The GloMax20 / 20 luminometer and GloMax luminometer had relatively low signals.

[0036] [Figure 8] Figures 8A-8C are plots generated from the data shown in Figure 6. The plots show that both the GloMax20 / 20 and GloMax were able to detect samples 21 (Figure 8A) and 26 (Figure 8B) as positive (signal / background ratio > 3.0 is positive) at 30 minutes of infection, however, sample 24 (Figure 8C) required 2 hours of infection to show a positive result. The results for the GloMax20 / 20 luminometer and the GloMax luminometer were similar.

[0037] [Figure 9] FIG. 9 shows data for detection of L. monocytogenes environmental sponge samples that were surface seeded and enriched for 24 hours.

[0038] [Figure 10] Figure 10 shows the detection of microorganisms in Salmonella-seeded turkey samples using the device. Signal was measured using three different luminometers: GloMax, 3M, and Hygiena.

[0039] [Figure 11]11A, 11B, and 11C show diagrams of one embodiment of a self-contained device system for detecting microorganisms, with a swab (FIG. 11A) or bead (FIG. 11B) inserted into a container containing three compartments. Each compartment is separated by a snap-action seal. The first compartment contains the phage, the second compartment contains the substrate, and the third compartment contains the medium. In some embodiments, the solid support is a swab. In some embodiments, the device does not contain the medium (FIG. 11C).

[0040] [Figure 12] 12A, 12B, and 12C show diagrams of one embodiment of a self-contained device system for detecting microorganisms, with a swab (FIG. 12A) or bead (FIG. 12B) inserted into a container containing three compartments. Each compartment is separated by a snap-action seal. The first compartment contains the phage, the second compartment contains the medium, and the third compartment contains the substrate. After incubation with the phage and the medium, the seal separating the second and third compartments can be broken. In some embodiments, the solid support is a swab. In some embodiments, the device does not contain the medium (FIG. 12C).

[0041] [Figure 13] Figures 13A, 13B, and 13C show diagrams of one embodiment of a self-contained device system for detecting microorganisms, with a swab (Figure 13A) or bead (Figure 13B) inserted into a container containing three compartments. Each compartment is separated by a snap-action seal. The first compartment contains the medium, the second compartment contains the phage, and the third compartment contains the substrate. In some embodiments, the solid support is a swab. In some embodiments, the device does not contain the medium (Figure 13C).

[0042] [Figure 14]Figures 14A, 14B, and 14C show diagrams of one embodiment of a self-contained device system for detecting microorganisms, with a swab (Figure 14A) or bead (Figure 14B) inserted into a container containing three compartments. Each compartment is separated by a snap-action seal. The first compartment contains the medium, the second compartment contains the phage, and the third compartment contains the substrate. The device has a stop-lock mechanism for stepwise mixing of the reagents. In some embodiments, the solid support is a swab. In some embodiments, the device does not contain the medium (Figure 14C).

[0043] [Figure 15] Figures 15A, 15B, and 15C show diagrams of one embodiment of a self-contained device system for detecting microorganisms, with a swab (Figure 15A) or bead (Figure 15B) inserted into a container containing three compartments. Each compartment is separated by a snap-action seal. The first compartment contains the medium, the second compartment contains the phage, and the third compartment contains the substrate. The device has a stop-lock mechanism for stepwise mixing of the reagents. In some embodiments, the solid support is a swab. In some embodiments, the device does not contain the medium (Figure 15C).

[0044] [Figure 16] Figure 16 shows a diagram of one embodiment of a self-contained device system for a system for detecting microorganisms, with a swab inserted into a container containing two compartments. Each compartment is separated by a snap action seal. The first compartment contains the phage and the second compartment contains the substrate. In some embodiments, the solid support is a swab.

[0045] [Figure 17] FIG. 17 shows a flow diagram of an embodiment utilizing a self-contained device system for the detection of microorganisms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Detailed Description of the Invention definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, 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 specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with the experimental procedures and techniques described herein are those well known and commonly used in the art.

[0047] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0048] As used herein, the terms "a," "an," and "the" may refer to one or more, unless specifically stated otherwise.

[0049] Use of the term "or" is used to mean "and / or," unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0050] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists from sample to sample.

[0051] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface to which a biomolecule can be attached. For example, a solid support can be an assay well (i.e., a microtiter plate or a multiwell plate, etc.), or a solid support can be a filter, an array, or a position on a mobile support such as a bead or a membrane (e.g., a filter plate or a lateral flow strip).

[0052] The term "label" or "indicator moiety" or "detectable moiety" or "detectable biomolecule" or "reporter" or "label" refers to a molecule that provides a signal that can be measured in a qualitative or quantitative assay. For example, an indicator moiety can include an enzyme that can be used to convert a substrate into a measurable product. An indicator moiety can be an enzyme (e.g., luciferase, HRP, or AP) that catalyzes a reaction that produces bioluminescence. Alternatively, an indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0053] As used herein, "bacteriophage" or "phage" includes one or more of the bacterial viruses. In this disclosure, the terms "bacteriophage" and "phage" include viruses such as mycobacteriophage (for TB and paraTB, etc.), mycophage (for fungi, etc.), mycoplasma phages, and any other term that refers to a virus that can invade and use living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic life forms to replicate itself. Here, "microscopic" means that the maximum dimension is one millimeter or less. Bacteriophage are viruses that have naturally evolved to use bacteria as a means of self-replication.

[0054] As used herein, "enrichment culture", "culturing for enrichment", "cultured for enrichment", or "enrichment culture" refers to traditional culturing, such as incubation in a medium favorable for microbial growth, and should not be confused with other possible uses of the term "enrichment", such as enrichment by removing liquid components of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not involve traditional promotion of microbial growth. In some embodiments of the methods described herein, enrichment cultures of short duration can be used, but if cultures are used, they do not require, and are much shorter than, traditional enrichment culture periods.

[0055] As used herein, "recombinant" refers to genetic (i.e., nucleic acid) modifications typically performed in a laboratory to combine genetic material not found in nature. This term is used interchangeably with the term "modified" herein.

[0056] As used herein, "RLU" refers to relative light units measured by a luminometer (e.g., GLOMAX® 96) or similar device that detects light. For example, detection of a reaction between luciferase and an appropriate substrate (e.g., the reaction of NANOLUC® with NANO-GLO®) is often reported in RLUs detected. overview

[0057] The present invention takes advantage of the high specificity of recombinant bacteriophages that are capable of binding with high affinity to specific microorganisms to detect the presence and / or amount of specific microorganisms in a sample.

[0058] Disclosed herein are compositions, methods, kits, and systems that demonstrate extraordinary sensitivity and speed of detection of microorganisms of interest in test samples (e.g., food samples, water samples, dairy samples, environmental samples, commercial samples, clinical samples, or other biological samples) using assays that do not use culture for enrichment or, in some embodiments, that perform a minimal incubation period during which the microorganisms can potentially grow. These compositions, methods, kits, and systems are capable of detecting microorganisms in a time frame that is shorter than previously thought possible.

[0059] The compositions, methods, kits, and system embodiments of the present invention can be applied to the detection of various microorganisms (e.g., bacteria, fungi, yeasts) in various environments, including, but not limited to, the detection of pathogens from food, water, dairy, environmental, commercial, clinical, or other biological samples. The recombinant bacteriophage-based detection embodiments disclosed herein can be adapted to any bacteria or other microorganism of interest (e.g., pathogenic microorganisms) for which a specific bacteriophage is available that does not recognize microorganisms not intended to be detected. The methods of the present invention provide high detection sensitivity and specificity without the need for rapid and traditional biological enrichment (e.g., culture). Thus, a variety of microorganisms can be detected using the methods of the present invention.

[0060] 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 various environments, including, but not limited to, detection of pathogens from food, water, dairy, environmental, commercial, clinical, or other biological samples. The methods of the present invention can rapidly provide high detection sensitivity and specificity without the need for traditional biological enrichment (e.g., culture), which is a surprising aspect since all available methods with the desired sensitivity and specificity require culture.

[0061] Also disclosed herein are systems and methods that use the device to detect microorganisms in a test sample (e.g., a food sample, a water sample, a dairy sample, an environmental sample, a commercial sample, a clinical sample, or other biological sample). The method uses a self-contained device that includes a solid support that can be used to collect the sample. The device further includes a first compartment that includes a bacteriophage having a genetic construct inserted into the bacteriophage genome, where the construct includes a promoter and an indicator gene. The method includes contacting the recombinant bacteriophage from the first compartment with the sample such that the recombinant bacteriophage infects one or more microorganisms in the sample, thereby producing an indicator gene product ("indicator"), and detecting the indicator. In some aspects, the device further includes a second compartment that includes a substrate specific for detecting the indicator. In some embodiments, the method further includes contacting the sample infected with the bacteriophage with the substrate, whereby the indicator is detected. In these embodiments, each compartment is separated from the immediately adjacent compartment by a snap-action seal that, when broken, allows the contents of the compartment to escape the compartment and mix with the sample or contents from the other compartments. For example, a user can break the snap-action seal so that recombinant bacteriophage from a first compartment contacts the sample on the solid support, thereby infecting the microorganisms bound on the solid support. Upon infection of the microorganism, the indicator gene is expressed to produce an indicator protein that can be detected by various detection devices. The presence of a signal indicates the presence of the microorganism in the sample.

[0062] The embodiments of the devices, compositions, methods, kits, and systems of the present invention can be applied to the detection of various microorganisms (e.g., bacteria, fungi, yeasts) in various environments, including, but not limited to, the detection of pathogens from food, water, dairy, environmental, commercial, clinical, or other biological samples. The detection embodiments disclosed herein can be adapted to any bacteria or other microorganism of interest (e.g., pathogenic microorganisms) for which specific recombinant bacteriophages that do not recognize other unintended microorganisms are available. The methods of the present invention provide high detection sensitivity and specificity without the need for rapid and traditional biological enrichment (e.g., culture). This is a surprising aspect, since all available methods with the desired sensitivity and specificity require culture. Detection of microorganisms in a sample using a self-contained device that houses the reagents required for microbial detection in separate compartments until the time of detection is convenient and efficient. The device is easy to use and does not require extensive training. sample

[0063] Each of the compositions, methods, kits, and system embodiments of the present invention allows for rapid detection and / or quantification of microorganisms in a sample, e.g., the methods of the present invention can be performed in a shorter period of time and with better results.

[0064] In certain embodiments, recombinant bacteriophages are used to detect microorganisms of interest. Microorganisms that can be detected by the compositions, methods, kits, and systems of the present invention include pathogens of commercial, medical, or veterinary concern. Any microorganism for which a recombinant bacteriophage specific for a particular microorganism has been identified can be detected by the method of the present invention. Those skilled in the art will understand that there are no limitations to the application of the method other than the availability of the necessary recombinant bacteriophage / microorganism pair.

[0065] Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food or water-borne pathogens. Bacterial cells detectable by the present invention include, but are not limited to, all species of Salmonella, all species of E. coli (including, but not limited to, E. coli O157:H7 (and other Shiga toxin- and enterotoxin-producing strains of E. coli)), all species of Listeria (including, but not limited to, 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, Brucella spp., Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Clostridium botulinum, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei, Yersinia spp., Vibrio spp. Staphylococcus aureus, and Streptococcus spp.

[0066] The sample may be an environmental sample, a food sample, or a water sample. The sample may be a medical or veterinary sample. The sample may be a liquid, solid, or semi-solid. The sample may be a swab of a solid surface. The sample may include environmental materials such as a water sample, or a filter from an air sample, or an aerosol sample from a cyclone. The sample may be a sample of beef, poultry, processed foods, milk, cheese, or other dairy products. Medical or veterinary samples include, but are not limited to, blood, sputum, cerebrospinal fluid, and fecal samples. In some embodiments, the sample may be various types of swabs.

[0067] In some embodiments, samples can be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples (including but not limited to milk and fruit juices) can be assayed directly. In other embodiments, samples can be diluted or suspended in a solution (which may include but is not limited to a buffer or bacterial culture medium). Samples that are solid or semi-solid can be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. In some embodiments, samples should be maintained within a pH range that promotes attachment of recombinant bacteriophage to host bacterial cells. In some embodiments, the preferred pH range may be a pH range appropriate for bacteriophage attached to bacterial cells. Samples should also be diluted or suspended in a solution of appropriate concentrations of divalent and monovalent cations (Na + , Mg 2+ , and K + (including but not limited to)

[0068] In some embodiments, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. During the process in which the bacteriophage are attaching to the bacterial cells, the sample may be maintained at a temperature that facilitates the activity of the bacteriophage. Such a temperature is at least about 25° C. and not more than about 45° C. In some embodiments, the sample is maintained at about 37° C. In some embodiments, the sample is subjected to gentle mixing or shaking during binding or infection of the recombinant bacteriophage. Methods for using recombinant bacteriophages for the detection of microorganisms

[0069] Methods for using recombinant bacteriophage to detect a microorganism of interest have been described previously. The assay may include a variety of appropriate control samples. For example, a control sample that does not contain recombinant bacteriophage and / or a control sample that contains recombinant bacteriophage but does not contain bacteria may be assayed as a control for background signal levels.

[0070] As noted herein, in certain embodiments, the invention may include methods of using recombinant bacteriophages to detect microorganisms. The methods of the invention may be embodied in a variety of ways.

[0071] In some aspects, the present invention includes a method for detecting a microorganism of interest. The method can use a recombinant bacteriophage for the detection of the microorganism of interest. For example, in certain embodiments, the microorganism of interest is a bacterium, and the recombinant bacteriophage is derived from a bacteriophage that specifically recognizes the bacterium of interest. In certain embodiments, the method can include detecting the bacterium of interest in a sample by incubating the sample with a plurality of recombinant bacteriophages that can bind to the bacterium of interest. The plurality of recombinant bacteriophages that bind to a single microorganism can be any number greater than 1, but preferably at least 5×10 4 pieces, or at least 1×10 5 pieces, or at least 1×10 6 pieces, or at least 1×10 8 pieces, or at least 1×10 9 pieces, or at least 1×10 10 It is a recombinant bacteriophage.

[0072] In certain embodiments, the recombinant bacteriophage comprises an indicator moiety. The method may include detecting the indicator moiety of the recombinant bacteriophage, where positive detection of the indicator moiety indicates that the bacterium of interest is present in the sample.

[0073] In some embodiments, the invention may include a method for detecting even as little as one microorganism of interest in a sample, the method comprising the steps of: incubating the sample with a plurality of recombinant bacteriophages that bind to the microorganism of interest, where the recombinant bacteriophages contain an indicator moiety and are contacted with the sample to be tested under conditions such that the plurality of recombinant bacteriophages bind to the bacteria of interest; separating unbound recombinant bacteriophages from cell-bound recombinant bacteriophages; and detecting the indicator moiety resulting from bacterial infection, where positive detection of the indicator moiety indicates the presence of the microorganism of interest in the sample. An embodiment includes incubating the sample with at least 5×10 8 pieces, or at least 5 × 10 9 pieces, or at least 5 × 10 10 pieces, or at least 5 × 10 11 pieces, or at least 5 × 10 12 pieces, or at least 5 × 10 13 The method may include incubating the recombinant bacteriophage with the

[0074] In some embodiments, the detection step will require the addition of a substrate for interaction with the indicator enzyme. The choice of a particular indicator is not critical to the invention, but the indicator will be capable of generating a detectable signal by itself, by the instrument, or in conjunction with one or more additional signal generating components (such as an enzyme / substrate signal generating system).

[0075] In certain embodiments, the assay may utilize recombinant bacteriophage to identify the presence of specific microorganisms. The assay may be modified to accommodate different sample types or sizes and assay formats. The recombinant bacteriophage-using embodiment of the present invention may be capable of rapidly detecting specific bacterial strains with total assay times of less than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12 hours, depending on the sample type, sample size, and assay format. For example, the time required may be somewhat shorter or longer, depending on the affinity of the recombinant bacteriophage and / or the type of bacteria to be detected in the assay, the type and size of the sample to be tested, the complexity of the physical / chemical environment, and the concentration of endogenous non-target bacterial contaminants.

[0076] FIG. 1 shows an embodiment of an assay for detecting bacteria of interest using recombinant bacteriophage according to an embodiment of the present invention. A wide variety of configurations and reagent combination steps are possible. In the embodiment shown here, an aliquot of indicator phage (or lyophilized indicator phage) is contained in the first compartment of the device. A swab containing bacteria is introduced into the first compartment and incubated for a period of time (e.g., 45 minutes at 37° C.) sufficient for the phage to replicate and produce a soluble indicator (e.g., luciferase). The seal between the first compartment containing the soluble indicator and phage and the second compartment can then be broken, allowing the soluble indicator protein to come into contact with a substrate contained in the second compartment. A luminometer can be used to detect the reaction of the indicator (e.g., luciferase) with the substrate. Experiments utilizing this method are described herein. In some embodiments, after infection, the contents of the device can be drawn back into the first compartment to mix the infected bacteria with the substrate. In a further embodiment, the contents can then be pushed back into the second compartment so that the indicator signal can be read with a luminometer.

[0077] In some embodiments, samples may be enriched prior to testing by incubation in conditions that promote growth. In such embodiments, the enrichment period may be 1, 2, 3, 4, 5, 6, 7 hours, or up to 8 hours or more, depending on the type and size of the sample.

[0078] Thus, in some embodiments, the indicator bacteriophage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Thus, the method may include detecting an indicator moiety produced during replication of the phage, where detection of the indicator indicates that the bacterium of interest is present in the sample.

[0079] In some embodiments, the indicator bacteriophage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Thus, the method may include detecting an indicator moiety produced during replication of the phage, where detection of the indicator indicates that the bacterium of interest is present in the sample.

[0080] As described in more detail herein, the methods and systems of the present invention may utilize a range of concentrations of parent indicator bacteriophage that will infect bacteria present in the sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly find, bind, and infect target bacteria (such as single cells) that are present in very low numbers in the sample. In some embodiments, the phage concentration may be sufficient to find, bind, and infect target bacteria in less than one hour. In other embodiments, these events may occur in less than two or three hours after the addition of the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubation step is greater than 1×10 5 >PFU / mL, 1×10 6 >1×10 PFU / mL 7 More than PFU / mL.

[0081] The method of the present invention may include various other steps to increase sensitivity. For example, as discussed in more detail herein, the method may include steps of capturing bacteria and washing the captured and bound bacteria to remove excess recombinant bacteriophage and increase the signal-to-noise ratio. In some embodiments, positive detection of the indicator moiety requires that the signal-to-background ratio generated by the detection of the indicator moiety is at least 2.0 or at least 2.5.

[0082] Figures 2-10 demonstrate data and plots from an exemplary embodiment of a recombinant bacteriophage assay.

[0083] Figures 2, 3A, and 3B relate to the detection of L. monocytogenes. Figure 2 shows data from the detection of L. monocytogenes cultures using one embodiment of a self-contained device with a swab as the solid support. Signals corresponding to the presence of bacteria were detected by a Hygiena luminometer, a GloMax luminometer, and a GloMax20 / 20 luminometer. Table 1 shows the results of log phase cultures and Table 2 shows the results of overnight cultures.

[0084] Figures 3A and 3B are plots generated from the data shown in Table 1. Figure 3A shows measurements of the signal detected using the Hygiena. Swabs were inoculated with log phase cells at the indicated CFU levels. Samples were immediately infected with Listeria phage cocktail for 4 hours. Substrate was added and samples were read in the Hygiena luminometer. A signal above 10 RLU was considered positive. Using this method, approximately 25,000 CFU are required to obtain a positive result.

[0085] FIG. 3B shows measurements of signal detected using a GloMax20 / 20 luminometer and a GloMax (also known as GloMax96) luminometer. Swabs were inoculated with log-phase cells at the indicated CFU levels. Samples were immediately infected with Listeria phage cocktail for 4 hours. Substrate was added and samples were read on either a GloMax20 / 20 luminometer (1 mL sample) or a GloMax (150 μl sample) luminometer. A signal / background ratio of >3.0 is considered positive. Using this method, approximately 5,000 CFU are required to obtain a positive result.

[0086] Figures 4-8 relate to embodiments demonstrating the detection of Salmonella. Figure 4 shows the results of detection of Salmonella in ground turkey meat inoculated with Salmonella. Table 3 shows the uninoculated control samples and Table 4 shows the uninoculated turkey samples. The test was repeated using various inoculations and infection times.

[0087] Figures 5A and 5B are plots made from the data in Figure 4. Figure 6A shows that turkey samples inoculated with Salmonella were detected as positive at every incubation and infection time tested. Turkey samples were grown at 41°C for 24 hours after inoculation and then tested using the methods disclosed in this application. For relative signal: 0 hour incubation, 2 hour infection > 1 hour incubation, 0.5 hour infection > 0 hour incubation, 0.5 hour infection. Additionally, a comparison of the RLU signals shows that the GloMax luminometer shows a significantly higher signal than the Hygiena luminometer.

[0088] Figure 5B shows that detection using the GloMax20 / 20 luminometer and the GloMax luminometer showed similar signal / background ratios for the same samples. The GloMax20 / 20 showed a larger signal (Figure 5A), but the background was significantly higher than the GloMax. Thus, when determining signal / background, the two luminometers perform similarly.

[0089] Figure 6 shows data for detection of Salmonella in three turkey samples (samples 21, 24, and 26) that were inoculated with Salmonella prior to assay using the self-contained device. Samples were infected for different times as indicated prior to detection of signal.

[0090] Figures 7A-7C are plots constructed from the data presented in Figure 6. Figures 7A-7C show the results of an experiment in which three inoculated ground turkey samples were enriched for 24 hours, swabbed, and assayed. Samples 24 (Figure 7B) and 26 (Figure 7C) showed no signal by the Hygiena handheld luminometer for the sample infected with phage for 30 minutes, but did show a signal for the sample infected for 2 hours. The GloMax20 / 20 luminometer and GloMax luminometer had relatively low signals.

[0091] Figures 8A-8C are plots generated from the data shown in Figure 6. The plots show that both the GloMax20 / 20 and GloMax were able to detect samples 21 (Figure 8A) and 26 (Figure 8B) as positive (signal / background ratio > 3.0 is positive) at 30 minutes of infection, however, sample 24 (Figure 8C) required 2 hours of infection to show a positive result. The results for the GloMax20 / 20 luminometer and the GloMax luminometer were similar.

[0092] FIG. 9 shows data for detection of L. monocytogenes environmental sponge samples that were surface seeded and enriched for 24 hours.

[0093] Figure 10 shows the detection of microorganisms in Salmonella-seeded turkey samples using the device. Signal was measured using three different luminometers: GloMax, 3M, and Hygiena.

[0094] Recombinant bacteriophage assays retain specificity over time. For example, in numerous embodiments of the development and utilization of recombinant bacteriophage assays over multiple years for E. coli, Cronobacter, Salmonella, Listeria, and S. aureus, no change in host specificity was observed. These embodiments include over 25 recombinant luciferase reporter or indicator bacteriophages. Furthermore, no loss or inactivation of the reporter or indicator gene (e.g., luciferase) has been detected in any of the recombinant phages described herein. To prevent mishandling, recombinant bacteriophages are stored and preserved in multiple aliquots. Although bacteriophages are inherently stable, titers may decrease to various degrees when stored for extended periods of time (e.g., at 4°C for months or years). This is generally believed to be due to loss of DNA from the particle; however, active bacteriophages are recovered by plating plaques to obtain higher titers. Specificity is verified using each recombinant bacteriophage preparation by infection of different bacterial strains known to be positive and negative.

[0095] In some embodiments of the method of testing a sample, the use of a large excess of recombinant bacteriophage requires the separation of any unbound bacteria or other larger components of the sample from the excess of unbound recombinant bacteriophage. This can be done in a number of different ways commonly known to those skilled in the art. The microbial cells can be separated by centrifugation, filtration by size, or selective immobilization. In some embodiments, filtration by size is performed by a filter well. In other embodiments, magnetic separation can be used for selective immobilization. For example, the sample can be filtered through a 0.45 μm or 0.22 μm membrane before or after incubation with the recombinant bacteriophage to capture the target microorganisms (e.g., bacteria) on the solid support. The captured microorganisms can then be washed one or more times on the solid support to ensure that only specifically bound recombinant bacteriophage remain. Alternatively, the microorganisms can be captured on microbeads or another solid surface using specific or non-specific binding mechanisms. Other formats for separating the components of the sample are also possible.

[0096] A variety of solid supports can be used. In certain embodiments, the solid support can include a multi-well plate, a filter, a bead, a lateral flow strip, a filter strip, a filter disk, a filter paper, or a thin membrane designed for culturing cells (e.g., PetriFilm from 3M). Other solid supports can also be suitable. For example, in some embodiments, the test sample microorganisms can be captured by binding to the surface of a swab, a plate, or by filtering the sample through a bacteriological filter (e.g., a spin filter or plate filter with a pore size of 0.45 μm). In one embodiment, the microorganisms captured on the filter or plate surface are incubated with recombinant bacteriophage, followed by one or more washes to remove excess unbound recombinant bacteriophage.

[0097] Alternatively, in some embodiments, the capture step may be based on other characteristics of the microorganism of interest, such as size. In embodiments utilizing size-based capture, the solid support may be a spin column filter. In some embodiments, the solid support comprises a 96-well filter plate. Alternatively, the solid support for capture may be a location on an array or a position on a mobile support, such as a bead.

[0098] In some embodiments, the solid support is coated with a cell-binding moiety that binds with high affinity to the microorganism of interest in the sample, allowing more bacteria to be bound to the solid support, increasing the sensitivity and specificity of the assay.

[0099] In one embodiment, when the microorganism of interest is a bacterium, the recombinant bacteriophage can bind to the bacterium via a cell-binding domain derived from the bacteriophage. For example, well-studied phages of E. coli include T1, T2, T3, T4, T5, T7, lambda; other E. coli phages available in the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. Salmonella phages include SPN1S, 10, ε15, SEA1, and P22. Listeria phages include LipZ5, P40, vB_LmoM_AG20, P70, and A511. Staphylococcus phages include P4W, virus K, Twort, φ11, 187, P68, and φWMY.

[0100] In some embodiments, samples may be enriched prior to testing by incubation in conditions that promote growth. In such embodiments, the enrichment period may be 1, 2, 3, 4, 5, 6, 7 hours, or up to 8 hours or more, depending on the type and size of the sample.

[0101] In other embodiments, the sample may be enriched after capture of the bacteria onto the solid support. In such embodiments, the enrichment period may be 1, 2, 3, 4, 5, 6, 7 hours, or up to 8 hours or more, depending on the type and size of the sample.

[0102] Thus, in some embodiments, the recombinant bacteriophage comprises a detectable indicator moiety, and binding to a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated by the indicator moiety. Thus, the method may comprise detecting the indicator moiety of the recombinant bacteriophage, where detection of the indicator indicates that the bacterium of interest is present in the sample.

[0103] In some embodiments of the method of the present invention, microorganisms can be detected without any isolation or purification of the microorganism from the sample.For example, in certain embodiments, a sample containing one or more target microorganisms can be directly applied to an assay vessel, such as a spin column, a microtiter well, or a filter, and assayed in said assay vessel.That is, the microorganisms are captured on a membrane with a pore size that is too small for the microorganism to pass through.Various embodiments of such assays are disclosed herein.

[0104] Aliquots of test samples may be dispensed directly into wells of a multi-well plate, recombinant bacteriophage may be added, and after a sufficient binding time for binding, the cells may be captured on a solid surface such as a plate, bead, or filter substrate such that excess unbound recombinant bacteriophage can be removed in one or more subsequent washing steps. A substrate for the indicator moiety (e.g., a luciferase substrate for a luciferase indicator) is then added and assayed for detection of the indicator signal. Some embodiments of the method may be performed on a filter plate. Some embodiments of the method may be performed with or without concentration of the sample prior to binding with the recombinant bacteriophage.

[0105] For example, in many embodiments, the assay is performed using a multi-well plate. The choice of plate (or any other container in which detection can be performed) can affect the detection process. For example, some plates may contain a colored or white background, which can affect the detection of luminescence. Generally, white plates are highly sensitive, but also have a high background signal. Plates of other colors may have a lower background signal, but are also slightly less sensitive. Furthermore, the background signal may be due to light leakage from one well to another adjacent well. Some plates have white wells and the remaining plates are black, so the signal inside the wells is high, while light leakage between wells is prevented. This combination of white wells with black plates can reduce the background signal. Thus, the choice of plate or other assay container can affect the sensitivity and background signal of the assay. In some embodiments, the detection of the microorganism of interest can be completed without the need for culturing the sample. For example, in certain embodiments, the total detection time required is less than 12.0 hours, less than 11.0 hours, less than 10.0 hours, less than 9.0 hours, less than 8.0 hours, less than 7.0 hours, less than 6.0 hours, less than 5.0 hours, less than 4.0 hours, less than 3.0 hours, less than 2.5 hours, less than 2.0 hours, less than 1.5 hours, less than 1.0 hour, less than 45 minutes, or less than 30 minutes. Minimizing the time to obtain results is important for food and environmental testing for pathogens.

[0106] In contrast to the assays known in the art, the method of the present invention can detect individual microorganisms. Thus, in certain embodiments, the method can detect 10 or less microbial cells (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 microorganisms) or 20 or less, or 30 or less, or 40 or less, or 50 or less, or 60 or less, or 70 or less, or 80 or less, or 90 or less, or 100 or less, or 200 or less, or 500 or less, or 1000 or less microbial cells present in a sample. For example, in certain embodiments, the recombinant bacteriophage is highly specific for S.Aureus, Listeria, Salmonella, or E. coli. In one embodiment, the recombinant bacteriophage can distinguish S.Aureus, Listeria, Salmonella, or E. coli in the presence of more than 100 other types of bacteria. In one embodiment, the recombinant bacteriophage can distinguish a specific serotype within a certain type of bacteria (e.g., E. coli O157:H7) in the presence of over 100 other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the recombinant bacteriophage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample.

[0107] Thus, aspects of the invention provide methods for detecting a microorganism in a test sample via a reporter or indicator moiety. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety may be associated with a recombinant bacteriophage. The indicator moiety may react with a substrate to generate a detectable signal or may generate an intrinsic signal (e.g., a fluorescent protein). A fluorescent protein naturally fluoresces by releasing energy as a photon when an electron-containing fluorescent moiety absorbs a photon (intrinsic or autofluorescence). A fluorescent protein (e.g., GFP) may be expressed as a fusion protein. In some embodiments, the detection sensitivity may reveal the presence of as few as 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in the test sample. In some embodiments, even a single cell of the microorganism of interest may provide a detectable signal.

[0108] The selection of a particular indicator moiety is not critical to the invention, but the indicator moiety will be capable of producing a detectable signal by itself, by the instrument, or in conjunction with one or more additional signal generating components (such as an enzyme / substrate signal generating system).Several recombinant bacteriophages can be formed by varying either the indicator moiety or the reporter of the recombinant bacteriophage; it will be recognized by one of skill in the art that the selection will include consideration of the microorganism to be detected and the desired means of detection.

[0109] For example, one or more signal-producing components can be reacted with an indicator moiety to generate a detectable signal. In some embodiments, the indicator can be a bioluminescent compound. If the indicator moiety is an enzyme, the detectable signal is amplified by reacting the enzyme with one or more substrates or additional enzymes and substrates to produce a detectable reaction product. In an alternative signal-producing system, the indicator can be a fluorescent compound that does not require enzymatic manipulation of the indicator to produce a detectable signal. Fluorescent molecules, including, for example, fluorescein and rhodamine and their derivatives and analogs, are suitable for use as indicators in such systems. In yet another embodiment, the indicator moiety can be a cofactor, which is then reacted with an enzyme and one or more substrates or additional enzymes and substrates to produce a detectable reaction product to amplify the detectable signal. In some embodiments, the detectable signal is colorimetric.

[0110] The detectable indicator moiety is an important feature of the recombinant bacteriophage and can be detected directly or indirectly. The indicator moiety provides a detectable signal by which the binding reaction can be monitored to provide a qualitative and / or quantitative measure. The relative amount and location of the signal produced by the modified or signaling microorganism can serve to indicate the presence and / or amount of the microorganism. The indicator moiety can also be used to select and isolate the modified or signaling microorganism, such as by using flow sorting or magnetic separation media.

[0111] In some embodiments, the indicator moiety of the recombinant bacteriophage may be detectable directly or after incubation with a substrate. Many different types of detectable biomolecules suitable for use as indicator moieties are known in the art and many are commercially available. In some embodiments, the recombinant bacteriophage includes an enzyme that serves as the indicator moiety. In some embodiments, the indicator or reporter of the recombinant bacteriophage encodes a detectable enzyme. The indicator moiety may emit light and / or be detectable by a color change. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), green fluorescent protein (GFP), or luciferase (Luc). In some embodiments, these enzymes may serve as indicator moieties. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0112] Thus, in some embodiments, the recombinant bacteriophage of the methods, systems, or kits is a wild-type bacteriophage that has been genetically modified with the sequence of an indicator protein (such as a fluorescent protein or a luciferase protein).

[0113] Bacteriophages are able to infect and lyse specific bacteria.

[0114] The detection of the indicator may include the detection of luminescence. In some embodiments, a luminometer may be used to detect the reaction of the indicator (e.g., luciferase) with the substrate. A luminometer can be used to detect RLU, or other machines or devices can be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other luminescence. Although absolute RLU is important for detection, a high signal-to-background ratio is also required (e.g., greater than 2.0, greater than 2.5, or greater than 3.0) to reliably detect single cells or a small number of cells.

[0115] In some embodiments, the reaction of the indicator moiety (e.g., luciferase) with the substrate may be allowed to continue for 30 minutes or more, and detection at various time points may be desirable to optimize sensitivity. For example, the luminometer can be read initially and then at 3 minute, or 5 minute, or 10 minute, or 15 minute intervals until the reaction is complete.

[0116] Thus, in some embodiments utilizing recombinant bacteriophages, the present invention includes a method for detecting a microorganism of interest comprising the steps of capturing at least one sample bacterium; incubating at least one bacterium with a plurality of recombinant bacteriophages; allowing a binding time to the target microorganism in the sample; and detecting an indicator moiety, wherein detection of the indicator moiety demonstrates the presence of the bacterium in the sample.

[0117] 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 bacteriological filter (e.g., a 0.45 μm pore size spin filter or plate filter). In one embodiment, recombinant bacteriophage is added to the sample captured directly on the filter in a minimal or moderate volume. In one embodiment, the microorganisms captured on the filter or plate surface are subsequently washed one or more times to remove excess unbound recombinant bacteriophage.

[0118] In some embodiments, an aliquot of a test sample containing bacteria can be applied to a spin column and washed to remove any excess bacteriophage after incubation with the recombinant bacteriophage, and the detected amount of indicator will be proportional to the amount of target bacteria present in the sample.

[0119] The indicator (e.g., luciferase) bound to the bacteria can then be measured and quantified. In one embodiment, the solution is spun through a filter and the filtrate is collected in a new container (e.g., in a luminometer) for assay after adding a substrate for the indicator enzyme (e.g., luciferase substrate). Alternatively, the indicator signal can be measured directly on the filter.

[0120] In one embodiment, the microorganism is a bacterium, and the recombinant bacteriophage comprises an indicator moiety derived from the bacteriophage. In one embodiment, the indicator moiety is luciferase. Thus, in another embodiment, an indicator substrate (e.g., a luciferase substrate) may be incubated with a portion of the sample that is retained on the filter or bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a regular 96-well plate), and the substrate reaction may be detected by placing the plate directly in a luminometer.

[0121] For example, in one embodiment, the invention may include a method for detecting a pathogenic bacterium of interest comprising the steps of: binding cells captured on a 96-well filter plate with a plurality of recombinant bacteriophages; washing away excess recombinant bacteriophages; and detecting an indicator (e.g., luciferase) by adding a substrate and measuring enzymatic activity directly in the 96-well plate, where detection of enzymatic activity indicates the presence of the bacterium of interest in the sample.

[0122] In another embodiment, the present invention may include a method for detecting a microorganism of interest (such as S. Aureus), comprising the steps of: binding a plurality of recombinant bacteriophages to cells in a solution or suspension in a 96-well plate; washing away unbound recombinant bacteriophages from the cells with bound recombinant bacteriophages; and detecting an indicator (e.g., luciferase) by adding a substrate and measuring enzyme activity directly in the 96-well plate, where detection of enzyme activity indicates that the microorganism of interest (such as S. Aureus) is present in the sample. In some embodiments, the microorganism of interest may be captured on a solid support, such as a bead or filter. This capture may be performed before or after incubation with the recombinant bacteriophage. In some embodiments, the capture step is not necessary.

[0123] In some embodiments, the solution or suspension can be a consumable test sample (such as vegetable wash). In some embodiments, the solution or suspension can be concentrated LB broth, trypsin / tryptone soy broth, peptone water, or vegetable wash fortified with nutrient broth. In some embodiments, the solution or suspension can be bacteria diluted in LB broth.

[0124] In some embodiments, the target microbial cells must be intact for proper detection, i.e., the cells do not need to be viable, but the cell wall must be structurally intact, and therefore it is desirable to minimize lysis prior to the detection step.

[0125] In some embodiments, an initial enrichment step of the sample is useful, i.e., to concentrate any microorganisms or other relatively large material in the sample and remove excess liquid. However, it is possible to perform the assay without an initial enrichment step. Some embodiments include an initial enrichment step, and in some embodiments, this enrichment step can shorten the enrichment incubation time. In other embodiments, no enrichment period is necessary.

[0126] Some embodiments of the test method may further include a confirmatory assay. A variety of assays are known in the art to confirm the initial result, which is usually carried out later. For example, the sample can be cultured (e.g., CHROMAGAR® / DYNABEADS® assay), PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm the initial result.

[0127] Food safety assay embodiments include sample preparation steps. Some embodiments may include an enrichment time. For example, depending on the type and size of the sample, an enrichment time of 1, 2, 3, 4, 5, 6, 7, or 8 hours may be required. After these sample preparation steps, a high concentration of recombinant bacteriophage containing a reporter or indicator can be bound in a variety of assay formats, such as the format shown in FIG. 1.

[0128] Embodiments of the food assay are capable of detecting a single pathogenic bacterium in samples sized according to industry standards, with the time to obtain a result being reduced by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, depending on the sample type and size.

[0129] Therefore, some embodiments of the present invention meet the need to amplify detectable signals indicating the presence of bacteria by using recombinant bacteriophage methods. In certain embodiments, even a single bacterium is detected. The principles applied herein can be applied to the detection of various microorganisms. In this way, embodiments of the present invention can greatly amplify signals even from a single microbial cell of interest.

[0130] Aspects of the invention exploit the high specificity of binding moieties capable of binding to specific microorganisms (such as recognition of the binding moieties of infectious agents) as a means to detect and / or quantitate specific microorganisms in a sample. In some embodiments, the invention exploits the specificity of bacteriophages.

[0131] Some embodiments of the present invention disclosed and described herein utilize the discovery that a single microorganism can produce a large amount of indicator after infection with recombinant bacteriophage.This principle allows the indicator signal from one or a few cells to be amplified based on the specific recognition of microorganisms.For example, even a single bacterial cell can be exposed to multiple recombinant bacteriophages, thereby amplifying the indicator signal so that a single bacterium can be detected.

[0132] The unprecedented speed and sensitivity of detecting microorganisms using recombinant bacteriophage is an unexpected result. In some embodiments, the method of the present invention requires less than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours to detect a microorganism of interest. These are shorter time frames than previously thought possible. In some embodiments, the method can detect as few as 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the bacterial target. In some embodiments, even a single bacterial cell can be detected. In further embodiments, the present invention includes systems (e.g., computer systems, automated systems, or kits) that include components for carrying out the methods disclosed herein and / or for using the recombinant bacteriophage described herein.

[0133] Existing protocols for detecting pathogenic bacteria in food are complex, expensive, slow, labor intensive, and prone to false positives. Furthermore, phage-based detection methods add the additional complexity of infectious reagents and regulatory concerns. Detection using recombinant bacteriophages specific for a given pathogen offers an effective, rapid, and simple alternative test method.

[0134] Infectious agents can be highly specific for certain types of organisms. For example, bacteriophages can be specific for a particular bacterial genus, such as Listeria. For example, A511 bacteriophage is specific for the genus Listeria. Alternatively, bacteriophages can be specific for a particular bacterial species, such as E. coli. For some bacterial types, bacteriophages can even recognize certain subtypes of organisms with high specificity. For example, CBA120 bacteriophage is highly specific for E. coli O157:H7, and φYeO3-12 bacteriophage is highly specific for Y. enterocolitica serotype O:3.

[0135] In some embodiments of the recombinant bacteriophage described herein, the recombinant bacteriophage can be derived from T7, T4, T4-like, ViI, ViI-like, AR1, A511, A118, A006, A500, PSA, P35, P40, B025, B054, A97, φSM101, φ3626, CBA120, SPN1S, 10, ε15, P22, LipZ5, P40, vB_LmoM_AG20, P70, A511, P4W, K, Twort, or SA97. The recombinant bacteriophage also includes an indicator moiety (such as a fluorescent moiety, a fluorescent protein, a bioluminescent protein, or an enzyme) that allows the recombinant bacteriophage to generate a signal. In the recombinant bacteriophage, various types of reporters can be inserted into the bacteriophage genome to serve as indicator moieties. In some embodiments, the recombinant bacteriophage contains an enzyme (such as luciferase) that is detectable only upon addition of an appropriate substrate. For example, luciferase, alkaline phosphatase, and other reporter enzymes react with an appropriate substrate to provide a detectable signal. Some embodiments of the recombinant bacteriophage contain wild-type luciferase or an engineered luciferase (such as NANOLUC®). Other embodiments contain a fluorescent protein or another reporter protein.

[0136] In another embodiment, bacteriophages, phages, mycobacteriophages (such as for TB and paraTB), mycophages (such as for fungi), mycoplasma phages, and any other viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic life forms can be studied or copied to target the microorganism of interest. For example, well-studied phages of E. coli include T1, T2, T3, T4, T5, T7, λ; other E. coli phages available in the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. Salmonella phages include SPN1S, 10, ε15, SEA1, and P22. Listeria phages include LipZ5, P40, vB_LmoM_AG20, P70, and A511. Staphylococcus phages include P4W, Virus K, Twort, φ11, 187, P68, and φWMY.

[0137] Some embodiments of the present invention exploit the binding specificity of recombinant bacteriophage for rapid and sensitive targeting to bind and facilitate detection of bacteria of interest.

[0138] In some embodiments, the recombinant bacteriophage is derived from a bacteriophage specific to the target bacterium of interest (such as T7, T4, or another similar phage). Also, the recombinant bacteriophage may be derived from T4-like, T7-like, ViI, ViI-like, AR1, A511, A118, A006, A500, PSA, P35, P40, B025, B054, A97, φSM101, φ3626, CBA120, SPN1S, 10, ε15, P22, LipZ5, P40, vB_LmoM_AG20, P70, A511, P4W, K, Twort, or SA97.

[0139] In some embodiments, a small indicator gene product may be desirable. OpLuc and NANOLUC® proteins are only about 20 kDa (with coding regions of approximately 500-600 bp), while FLuc is about 62 kDa, requiring approximately 1,700 bp of coding region. For comparison, the T7 genome is approximately 40 kbp, while the T4 genome is approximately 170 kbp.

[0140] In addition, the indicator should generate a high signal-to-background ratio and be easily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO®, NANOLUC® in combination with an imidazopyrazinon substrate (furimazine) can generate a robust signal with low background.

[0141] In some recombinant bacteriophage embodiments, the indicator moiety can be any of a variety of biomolecules. The indicator can be a detectable product or an enzyme that produces a detectable product or a cofactor for an enzyme that produces a detectable product. In some embodiments, the indicator moiety of the recombinant bacteriophage is a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). For example, in one embodiment, the indicator is a luciferase enzyme. Various types of luciferases can be used. In another embodiment, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase, as described in more detail herein. In some embodiments, the luciferase is derived from Oplophorus. In some embodiments, the indicator is a genetically modified luciferase (such as NANOLUC®). Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties. In some embodiments, these enzymes may serve as indicator moieties.

[0142] The compositions of the invention may include one or more recombinant bacteriophages derived from one or more wild-type infectious agents (e.g., bacteriophages) and one or more indicator moieties. In some embodiments, the compositions may include a cocktail of different recombinant bacteriophages that may generate the same or different indicator signals. That is, a composition for detecting a microorganism may include all the same or different recombinant bacteriophages.

[0143] The previously described assays utilize recombinant bacteriophage that recognize and bind to specific bacteria that can be used. Recombinant bacteriophage assays can be performed in traditional laboratory settings or devices, such as the devices further described herein. Recombinant bacteriophage

[0144] As described in more detail herein, the device, method, system, and kit of the present invention includes a recombinant bacteriophage for use in detecting a microorganism of interest. The recombinant bacteriophage is included in a first compartment of the device disclosed above. In some embodiments, the present invention may include a composition including a recombinant bacteriophage having an indicator gene integrated into the genome of the bacteriophage. In some embodiments, the indicator gene is operably linked to a promoter that is not the native promoter of the bacteriophage. In some embodiments, the indicator gene is operably linked to a native promoter of the bacteriophage. A recombinant bacteriophage that includes an indicator gene or reporter gene is also referred to as an indicator bacteriophage in this disclosure.

[0145] The recombinant bacteriophage can include a reporter gene or indicator gene. In certain embodiments of the bacteriophage, the indicator gene does not code for a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host cell results in a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene region of the bacteriophage. Late genes code for structural proteins and are therefore generally expressed at higher levels than other phage genes. The late gene region can be a class III gene region and can include the gene for the major capsid protein.

[0146] Some embodiments involve the design (and optional preparation) of sequences for homologous recombination downstream of the major capsid protein gene. Other embodiments involve the design (and optional preparation) of sequences for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequences include a codon-optimized reporter gene followed by an untranslated region. The untranslated region may include a phage late gene promoter and a ribosome entry site.

[0147] In some embodiments, the indicator bacteriophage is derived from A511, P100, Listeria phage LMTA-94, LMA4, LMA8, T7, T4, or another similar phage. Also, the indicator bacteriophage may be a P100 virus, T4-like, T7-like, ViI, ViI-like, Cronobacter, Salmonella, Listeria, or Staphylococcus-specific bacteriophage, or a T7, T7-like, T4, T4-like, P100 virus, Cronobacter, Salmonella, Listeria, or Staphylococcus-specific bacteriophage. The indicator phage may be derived from another bacteriophage having a genome that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the bacteriophage ViI or ViI-like (or Vi1 virus-like, according to GenBank / NCBI) bacteriophage. In some embodiments, the indicator phage is derived from a bacteriophage that exhibits high specificity for a particular pathogenic microorganism. The genetic modification can avoid the deletion of wild-type genes, and thus the modified phage may retain more similarity to the wild-type bacteriophage than many commercially available phages. Bacteriophages from the environment may be more specific to bacteria found in the environment and therefore may be genetically different from commercially available phages.

[0148] Furthermore, phage genes that are considered non-essential may have unrecognized functions. For example, an apparently non-essential gene may have important functions that increase the number of releases, such as slight cleavage, adaptation, or trimming functions of the assembly. Therefore, deleting a gene to insert an indicator may be detrimental. Most phages can package DNA that is a few percent larger than their native genome. With this in mind, a smaller indicator gene may be a better choice to engineer bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (coding region about 500-600 bp), while FLuc is about 62 kDa (coding region about 1,700 bp). For comparison, the genome of T7 is roughly 40 kbp, while the T4 genome is about 170 kbp and the genome of bacteriophages specific for Cronobacter, Salmonella, or Staphylococcus is about 157 kbp. In addition, the reporter gene should not be endogenously expressed by the bacteria (it is not part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO®, NANOLUC® in combination with an imidazopyrazinon substrate (furimazine) can generate a robust signal with low background.

[0149] In some indicator phage embodiments, the indicator gene can be inserted into a non-translated region to avoid disruption of a functional gene, thereby leaving the wild-type phage gene intact and enhancing fitness when infecting non-laboratory strains of bacteria. Additionally, including stop codons in all three reading frames can help increase expression by reducing read-through, also known as leaky expression. This strategy can also eliminate the possibility of making low levels of fusion proteins that may appear as background signals (e.g., luciferase) that cannot be separated from the phage.

[0150] The indicator gene may express various biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. In another embodiment, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or engineered luciferase, as described in more detail herein. In some embodiments, the luciferase gene is from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene (such as NANOLUC®).

[0151] Thus, in some embodiments, the present invention includes genetically modified bacteriophages that contain a non-bacteriophage indicator gene within the late (class III) gene region. In some embodiments, the modified indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels like the viral capsid protein, but is not shut down like endogenous bacterial genes or even earlier viral genes.

[0152] In some embodiments, the late promoter is a P100 virus, T4-, T7-, or ViI-like promoter, or another phage promoter similar to a promoter found in the selected wild-type phage (i.e., not genetically modified). The late gene region may be a class III gene region, and the bacteriophage may be derived from a T7, T4, T4-like, ViI, ViI-like, Cronobacter, Salmonella, Staphylococcus, Listeria, or S. aureus-specific bacteriophage, or another naturally occurring bacteriophage having a genome at least 70, 75, 80, 85, 90, or 95% homologous to a T7, T4, T4-like, ViI, ViI-like, or Cronobacter, Salmonella, Staphylococcus, Listeria, or S. aureus-specific bacteriophage.

[0153] The genetic modification to the bacteriophage may include the insertion, deletion, or replacement of a small fragment of nucleic acid, a substantial portion of a gene, or an entire gene. In some embodiments, the inserted or replaced nucleic acid comprises a modified sequence. A modified indicator gene can be inserted into the bacteriophage genome so that it is under the control of a bacteriophage promoter. In some embodiments, the modified indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification can be configured so that the indicator protein product does not contain a polypeptide of a wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present invention includes a method for detecting a bacterium of interest, comprising incubating a test sample with such a recombinant bacteriophage.

[0154] In some embodiments, expression of the indicator gene in the progeny bacteriophage after infection of the host bacterium results in a free soluble protein product. In some embodiments, the modified indicator gene is not adjacent to the gene encoding the structural phage protein and therefore does not produce a fusion protein. Unlike systems that use fusion of a detection moiety to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., a soluble luciferase). In some embodiments, the indicator or reporter ideally does not include a bacteriophage structure. That is, the indicator or reporter is not attached to a phage structure. As such, the indicator or reporter gene is not fused to other genes in the recombinant phage genome. This can greatly improve the sensitivity of the assay (down to a single bacterium) and simplify the assay, which allows the assay to be completed in less than an hour for some embodiments, as opposed to the several hours required for further purification steps of constructs that produce detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding constraints that may alter the conformation of the enzyme active site or access to substrates.

[0155] Furthermore, fusion proteins, by definition, limit the number of moieties that can be attached to a protein subunit in a bacteriophage. For example, using a commercially available system designed to serve as a platform for fusion proteins, it is believed that approximately 415 copies of the fusion moiety can be obtained, which corresponds to approximately 415 copies of the gene 10B capsid protein in each T7 bacteriophage particle. Without this limitation, infected bacteria can be expected to express many more copies of the detection moiety (e.g., luciferase) than can be mounted on the bacteriophage. Furthermore, large fusion proteins (such as capsid-luciferase fusions) can inhibit the assembly of bacteriophage particles, resulting in the generation of fewer bacteriophage progeny. Thus, a soluble, non-fused indicator gene product may be preferred.

[0156] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may serve as indicator moieties. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0157] In some embodiments, the use of soluble detection moieties obviates the need to remove contaminating parental phage from lysates of infected sample cells. With a fusion protein system, any bacteriophage used to infect the sample cells will have a detection moiety attached to it and will be indistinguishable from daughter bacteriophages that also contain the detection moiety. Because detection of sample bacteria relies on detection of newly created (de novo synthesized) detection moieties, the use of fusion constructs requires an additional step of separating the old (parental) moieties from the newly created (daughter bacteriophage) moieties. This can be accomplished by washing infected cells multiple times prior to completion of the bacteriophage life cycle, inactivating excess parental phage after infection by physical or chemical means, and / or chemically modifying the parental bacteriophage with a binding moiety (such as biotin) that can then be bound and separated (such as by streptavidin-coated sepharose beads). However, even if all these attempts are made to remove them, if a high concentration of parent phage is used to ensure infection of a small number of sample cells, the parent phage may remain and generate background signals that may obscure the detection of signals from progeny phage in infected cells.

[0158] In contrast, soluble detection moieties expressed in some embodiments of the invention do not require purification of the parent phage from the final lysate, since the parent phage does not attach to any detection moiety. Thus, any detection moiety present after infection must have been created de novo and indicates the presence of infected bacteria. To take advantage of this advantage, the production and preparation of the parent phage may include purification of the phage from any free detection moiety produced during the production of the parent bacteriophage in bacterial culture. Some embodiments of the phage of the invention may be purified using standard bacteriophage purification techniques, such as sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, and dialysis, or derived technologies (such as Amicon brand concentrators-Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation may be used as part of the preparation of the recombinant phage of the invention to separate the parent phage particles from the contaminating luciferase protein produced during the growth of the phage in the bacterial host. In this way, the parent recombinant bacteriophage 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 observed when the recombinant bacteriophage are incubated with a test sample.

[0159] In some embodiments of the modified bacteriophage, the late promoter (e.g., class III promoter from Listeria-specific phage, T7, T4, or ViI) has high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes of the structural proteins that are assembled into the bacteriophage particle. These proteins are the most abundant proteins made by the phage, as each bacteriophage particle contains tens or hundreds of copies of these molecules. The use of viral late promoters can ensure optimal high-level expression of the luciferase detection moiety. The use of T4, T7, or ViI late promoters with late viral promoters, T4, T7, or ViI-based systems that are derived from, specific for, or active in the original wild-type bacteriophage from which the indicator phage is derived (e.g., Listeria-specific phage), can further ensure optimal expression of the detection moiety. The use of standard bacterial (non-viral / non-bacteriophage) promoters can, in some cases, be detrimental to expression, since these promoters are often downregulated during bacteriophage infection (because the bacteriophage prefers bacterial sources for phage protein production). Thus, in some embodiments, phages are preferably engineered to encode and express high levels of soluble (free) indicator moieties using arrangements in the genome that do not limit expression to the subunit number of phage structural components.

[0160] The compositions of the invention may include one or more wild-type or genetically modified bacteriophages (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may include a cocktail of different indicator phages that can encode and express the same or different indicator proteins. In some embodiments, the cocktail of bacteriophages includes at least two different types of recombinant bacteriophages. Device or apparatus for detecting microorganisms

[0161] Embodiments of the invention relate to methods of detecting a microorganism of interest using a self-contained device. In some embodiments, the device includes a solid support that can be used to collect a sample containing the microorganism of interest. In some embodiments, the device of the invention includes a tube with separate compartments in a continuous or branched arrangement (e.g., "ears" on a tube). The device may include several compartments, which can be configured for mixing and timing of various reagents of the method steps. In some embodiments, the device includes at least two compartments. In some embodiments, the top or upper compartment of the tube contains the recombinant bacteriophage, and a substrate compartment is present below the bacteriophage compartment. In some embodiments, the tube contains a growth medium.

[0162] In some embodiments, the device comprises at least two compartments, including a first compartment containing a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, said construct comprising a promoter and a reporter (indicator) gene; an entry hole in the first compartment for adding at least a portion of a test sample to the recombinant bacteriophage; and a second compartment containing a substrate or developer by which the reporter (indicator) gene can be detected.

[0163] In one embodiment, the first compartment contains the recombinant bacteriophage and the second compartment contains the substrate or revelation reagent. In some such embodiments, both reagents are mixed with the sample simultaneously. In other embodiments, the sample is added to the first compartment first to initiate infection. After a period of time, a conduit leading to the second compartment is opened and the substrate or revelation reagent is added to the infected sample and mixed.

[0164] In another embodiment, the first compartment contains a lyophilized infectious agent (e.g., a lyophilized recombinant bacteriophage) and the second compartment contains a substrate or revelation reagent. In some such embodiments, both reagents are mixed with the sample simultaneously (optionally capturing microorganisms from the sample on a solid support). In other embodiments, the sample is added to the first compartment first. After a period of time, a conduit leading to the second compartment is opened and a substrate or revelation reagent is added to the infected sample and mixed.

[0165] In some embodiments, a seal separates adjacent compartments, and the user breaks the seal(s), and both the substrate and phage encounter the swab simultaneously. Luciferase is produced and reacts with the substrate, thereby producing a detectable signal.

[0166] In some embodiments, the substrate compartment may be located between the medium compartment and the recombinant bacteriophage compartment. In other embodiments, the device does not include a medium compartment and the device does not include medium. Both reagents can be applied simultaneously when the user breaks the seal and the reagents are applied to the sample captured on the solid support.

[0167] In another embodiment, the substrate compartment may be located between the medium compartment and the recombinant bacteriophage compartment. In other embodiments, the device does not include a medium compartment and the device does not include medium. Both reagents can be applied simultaneously when the user breaks the seal and the reagents are applied to the sample captured on the solid support.

[0168] In some embodiments, a solid support is added to the tube, where the substrate or developer is at the bottom of the tube; the reaction between the enzyme and the substrate or between other reporters and pairing reagents is initiated by pressing down on the solid support. For ease of handling, the solid support can be attached to a stem.

[0169] In some embodiments, the solid support comprises a bead coated with a molecule (e.g., an antibody or cell-binding protein) that specifically captures the microorganism of interest, in other embodiments, the solid support comprises a swab or another non-specific capture mechanism.

[0170] Alternatively, in any of the embodiments described herein, the device does not contain culture medium.

[0171] The following figures provide illustrations of embodiments of the present invention.

[0172] In one embodiment, the present disclosure provides a self-contained microbial detection device system. Figures 11A, 11B, and 11C show an embodiment of a device 100. The device includes a solid support 16 and a container that includes three compartments. Each of the compartments is separated by a snap-action seal. The first compartment 10 contains the phage, the second compartment 12 contains the substrate, and the third compartment 14 contains the medium. The device is capable of incubating the phage and the substrate simultaneously with the sample. In some embodiments, the device does not contain the medium (Figure 11C).

[0173] Figures 12A, 12B, and 12C show a second embodiment of the device 200. The device includes a solid support 26 and a container that includes three compartments. Each of the compartments is separated by a snap action seal. The first compartment 20 contains the phage, the second compartment 22 contains the medium, and the third compartment 24 contains the substrate. In an embodiment, using the device shown in Figure 12A, the sample is first incubated with the phage before incubation with the substrate. In a further embodiment, using the device shown in Figure 12B, the solid support is immersed in the medium before collection of the sample. In some embodiments, the device does not contain the medium (Figure 12C).

[0174] Figures 13A, 13B, and 13C show a third embodiment of the device 300. The device includes a solid support 36 and a container that includes three compartments. Each of the compartments is separated by a snap action seal. The first compartment 30 includes the medium, the second compartment 32 includes the phage, and the third compartment 34 includes the substrate. In an embodiment, using the device shown in Figure 13A, the sample is first incubated with the phage before incubation with the substrate. In a further embodiment, using the device shown in Figure 13B, the solid support is dried before collection of the sample. In some embodiments, the device does not include the medium (Figure 13C).

[0175] 14A, 14B, 14C show a fourth embodiment of the device 400. The device includes a solid support 46 and a container containing three compartments. Each of the compartments is separated by a snap action seal. The first compartment 40 contains the medium, the second compartment 42 contains the phage, and the third compartment 46 contains the substrate. The device has a stop-lock mechanism for stepwise mixing of the reagents. In an embodiment, using the device shown in FIG. 14A, the sample is first incubated with the phage before incubation with the substrate. In a further embodiment, using the device shown in FIG. 14B, the solid support is immersed in the medium before collection of the sample. In some embodiments, the device does not contain the medium (FIG. 14C).

[0176] 15A, 15B, and 15C show a fifth embodiment of the device 500. The device includes a solid support 56 and a container containing three compartments. Each of the compartments is separated by a snap action seal. The first compartment 50 contains the medium, the second compartment 52 contains the phage, and the third compartment 54 contains the substrate. The device has a stop-lock mechanism for stepwise mixing of the reagents. In an embodiment, using the device shown in FIG. 15A, the sample is first incubated with the phage before incubation with the substrate. In a further embodiment, using the device shown in FIG. 15B, the solid support is dried before collection of the sample. In some embodiments, the device does not contain the medium (FIG. 15C).

[0177] Figure 16 shows a sixth embodiment of a device 600. The device includes a solid support 64 and a container containing two compartments. Each of the compartments is separated by a snap action seal. The first compartment 60 contains the phage and the second compartment 62 contains the substrate. In an embodiment, using the device shown in Figure 16, the sample is first incubated with the phage prior to incubation with the substrate.

[0178] FIG. 17 shows an embodiment of a method for detecting a microorganism, comprising the steps of (i) enriching a sample overnight, (ii) using a solid support from a self-contained device to collect the overnight enriched sample, (iii) infecting the sample with phage contained within a compartment of the self-contained device, and (iv) detecting the presence of the microorganism by reading / detecting a signal produced by the infection step.

[0179] In some embodiments, compartments included in protrusions or branches from a central tube allow for mixing of reagents from more than two directions, for example in the form of "ears". For example, two squeeze valves can be used to add medium and phage sequentially or simultaneously to the main compartment, or to add other reagents. Different reagents can be added and mixed in larger adjacent compartments by varying the positioning of other compartments relative to the central compartment. Method of using a device for the detection of microorganisms

[0180] As described herein, in certain embodiments, the invention may include a method of detecting a microorganism, comprising contacting a sample with a solid support, such as contacting a recombinant bacteriophage from a first compartment with a microorganism captured on a solid support by breaking a snap action seal of the first compartment, such that the microorganism is captured on the solid support. During and / or after infection, the bacteriophage expresses an indicator gene to produce an indicator, which can be detected by various detection devices. In some embodiments, detection of the indicator may require the addition of a substrate that reacts with the indicator to produce a detectable signal. The presence of a signal indicates the presence of the microorganism in the sample.

[0181] In another embodiment, the present invention may include a method of detecting a microorganism, comprising contacting a sample with a solid support, such as contacting a recombinant bacteriophage from a first compartment with a microorganism captured on a solid support by breaking a snap action seal of the first compartment, such that the microorganism is captured on the solid support. The indicator portion of the recombinant bacteriophage can be detected by a variety of detection devices. In some embodiments, detection of the indicator may require the addition of a substrate that reacts with the indicator to produce a detectable signal. The presence of a signal indicates the presence of the microorganism in the sample. Sampling

[0182] In some embodiments, samples can be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples (including but not limited to milk and fruit juice) can be assayed directly. In other embodiments, samples can be diluted or suspended in a solution (which may include but is not limited to a buffer or bacterial culture medium). Samples that are solid or semi-solid can be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. In some embodiments, samples should be maintained within a pH range that promotes attachment of phage to host bacterial cells. In some embodiments, the preferred pH range may be a pH range appropriate for bacteriophage attached to bacterial cells. Samples should also contain appropriate concentrations of divalent and monovalent cations (including but not limited to Na+, Mg2+, and K+).

[0183] Preferably, throughout the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. During the process in which the bacteriophage are attaching to the bacterial cells, it is preferred to maintain the sample at a temperature that facilitates bacteriophage activity. Such a temperature is at least about 25° C. and not more than about 45° C. In some embodiments, the sample is maintained at about 37° C. In some embodiments, the sample is subjected to gentle mixing or shaking during bacteriophage binding or attachment.

[0184] The assay may include a variety of appropriate control samples, for example, a control sample that does not contain bacteria (e.g., a food sample) may be assayed as a control for background signal levels.

[0185] Sampling can be performed using a variety of methods. In some embodiments, the sample (e.g., a food sample) is first liquefied and a solid support (e.g., a solid support or beads) is immersed in the liquid sample. In some embodiments, the solid support is first immersed in culture medium in a tube before sampling. In some embodiments, the solid support is dried before sampling. In some embodiments, the liquid sample is first cultured for a period of time ("culture enrichment") (e.g., less than 24 hours, less than 12 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours of enrichment period).

[0186] In other embodiments, the sample may be enriched after the capture of microorganisms on the solid support. In some embodiments, the solid support with microorganisms can be incubated in a growth medium in the third compartment of the device to expand the number of microorganisms. This step is called incubation enrichment. In such embodiments, the enrichment period can be 1, 2, 3, 4, 5, 6, 7 hours, or up to 8 hours or more, depending on the type and size of the sample.

[0187] In some embodiments of the methods of the present invention, microorganisms can be detected without any isolation or purification of the microorganism from the sample. For example, in certain embodiments, a sample containing one or more microorganisms of interest can be applied directly to a solid support and the assay is performed within the device. infection

[0188] The methods disclosed herein include operating the device to contact the recombinant bacteriophage with the microorganism of interest. Upon contact of the microorganism, the bacteriophage replicates and expresses the indicator or reporter gene. See the "Recombinant Bacteriophage" section. The infection time (i.e., the time between when the sample is first contacted with the bacteriophage and when the substrate is added to the mixture) can vary depending on the type of bacteriophage and the concentration of the microorganism in the sample. The use of a device that captures bacteria on a solid support can significantly reduce the time required for infection, for example, the infection time can be 1 hour or less, while in standard assays, when a solid support is not used to capture bacteria, the infection is typically at least 4 hours, and in certain embodiments, the infection times disclosed herein are less than 6.0 hours, less than 5.0 hours, less than 4.0 hours, less than 3.0 hours, less than 2.5 hours, less than 2.0 hours, less than 1.5 hours, less than 1.0 hours, less than 45 minutes, or less than 30 minutes. In some embodiments, the infection time is about 1 hour, about 2 hours, or about 3 hours. Signal generation

[0189] The indicator produced by the expression of the indicator gene can be detected using methods well known to those skilled in the art. For example, one or more signal-producing components can be reacted with the indicator to generate a detectable signal. In some embodiments, the indicator can be a bioluminescent compound. If the indicator is an enzyme, the detectable signal is amplified by reacting the enzyme with one or more substrates or additional enzymes and substrates to produce a detectable reaction product. In an alternative signal-producing system, the indicator can be a fluorescent compound that does not require enzymatic manipulation of the indicator to produce a detectable signal. Fluorescent molecules, including, for example, fluorescein and rhodamine and their derivatives and analogs, are suitable for use as indicators in such systems. In yet another embodiment, the indicator moiety can be a cofactor, which is then reacted with an enzyme and one or more substrates or additional enzymes and substrates to produce a detectable reaction product to amplify the detectable signal. In some embodiments, the detectable signal is colorimetric. While the selection of a particular indicator is not critical to the invention, it should be noted that the indicator may generate a detectable signal by itself, by the instrument, or in conjunction with one or more additional signal generating components (such as an enzyme / substrate signal generating system).

[0190] In some embodiments, the detection step will require the addition of a substrate to interact with the indicator enzyme. The substrate can be added in a variety of ways. In some embodiments, the substrate is contained in a second compartment of the device, and breaking the snap action seal simultaneously contacts the phage (in the first compartment of the device) and the substrate with the microorganism (captured on the solid support). See FIG. 12. In some embodiments, the snap action seals are broken sequentially to contact the microorganism with the bacteriophage before contacting the substrate. See FIGS. 13A and 13B. In some embodiments, the method includes manipulating the stop lock to allow stepwise mixing such that the microorganism contacts the bacteriophage before contacting the substrate.

[0191] In some embodiments, the reaction of the indicator (e.g., luciferase) with the substrate may continue for 30 minutes or more, and it may be preferable to detect at various time points to optimize sensitivity. In some embodiments, the luminometer may be read initially and then at 3 minute, or 5 minute, or 10 minute, or 15 minute intervals until the reaction is complete. Signal detection

[0192] Detection of the signal produced by the indicator may include detection of luminescence. In some embodiments, the compartment of the device where the substrate is mixed with the test sample is transparent so that any signal resulting from infection and subsequent incubation with the substrate can be viewed. In this case, the signal can be detected through the wall of the compartment. In some embodiments, the device containing the reacted sample is inserted into the device to detect the resulting signal. In other embodiments, the detection device is used to scan the device containing the reacted sample.

[0193] In some embodiments, a luminometer (e.g., GloMax® 20 / 20 and GloMax® from Promega, Madison, WI) may be used to detect the indicator (e.g., luciferase). In some embodiments, a spectrophotometer, CCD camera, or CMOS camera may be used to detect color changes and other luminescence. While absolute RLU is important for detection, a high signal-to-background ratio is also required (e.g., greater than 2.0, greater than 2.5, or greater than 3.0) to reliably detect single cells or small numbers of cells. Background signals can be obtained by measuring control samples without microorganisms using the same procedure as above. In some embodiments, detection of signals from reporter or indicator genes may include the use of instruments that use, for example, photodiode or PMT (photomultiplier tube) technology. In some embodiments, a handheld luminometer may be used for signal detection. Suitable PMT handheld luminometers are available from 3M (Maplewood, MN), BioControl (Seattle, WA), and Charm Science (Lawrence, MA). Suitable photodiode handheld luminometers are available from Hygiena (Camarillo, CA) and Neogen (Lansing, MI). These handheld luminometers typically provide much lower readings compared to traditional luminometers (GloMax or GloMax20 / 20) for the same samples. As shown in the examples, multiple experiments show that the signal produced by the reaction was sufficient to be detected by these handheld luminometers. The assay was repeated multiple times using different types of microorganisms, including L. monocytogenes and Salmonella, with similar results each time. This indicates that the detection method using the aforementioned apparatus is sensitive and robust. Additionally, the ability of these handheld devices to detect microorganisms provides convenience and adaptability that is often lacking when using detection methods using traditional non-handheld detection devices. Systems and kits of the present invention

[0194] In some embodiments, the present invention includes a system (e.g., an automated system) or kit that includes components for carrying out the methods disclosed herein. In some embodiments, an apparatus is included in the system or kit of the present invention. Also, the methods described herein may utilize such a system or kit. Some embodiments described herein are particularly suitable for automation and / or kits, given the minimal amount of reagents and materials required to carry out the methods. In certain embodiments, each of the components of the kit may include a self-contained unit that can be delivered from a first location to a second location.

[0195] In some embodiments, the present invention includes a system or kit for rapid detection of a microorganism of interest in a sample. The system or kit may, in certain embodiments, include the above-described device and a signal detection component, where the aforementioned signal detection component is capable of detecting an indicator gene product produced from recombinant bacteriophage infection of the sample. In some embodiments, the signal detection component is a handheld device. In some embodiments, the signal detection component is a handheld luminometer.

[0196] Thus, in certain embodiments, the present invention may comprise a system or kit for rapid detection of a microorganism of interest in a sample, comprising a device comprising a first compartment containing a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, said construct comprising a promoter and an indicator gene. The system or kit may further comprise a second compartment containing a substrate and / or a third compartment containing a medium. One or more of these compartments are sealed and separated from the rest of the device by a snap action seal, which, when broken, releases the contents from the compartment and mixes with the sample.

[0197] In some embodiments, the system may include components for isolating the microorganism of interest from other components in the sample.

[0198] In some systems and / or kits, the same components can be used for multiple steps. In some systems and / or kits, the steps are automated, controlled by a user via computer input, and / or a liquid handling robot performs at least one step. In computerized systems, the system can be fully automated, semi-automated, or directed by a user via a computer (or some combination of these).

[0199] These systems and kits of the invention include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of devices suitable for carrying out the recited methods. The components need not be connected or arranged together in any particular manner. The invention includes any suitable arrangement of the components with respect to each other. For example, the components need not be in the same space. However, in some embodiments, the components are connected together in an integrated unit. In some embodiments, the same component can perform multiple functions. Computer system and computer readable medium

[0200] In certain embodiments, the present invention may include a system. The system may include at least some of the compositions of the present invention. The system may also include at least some of the components for carrying out the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present invention may include a system for rapid detection of a microorganism of interest in a sample. The system may include at least some of the compositions of the present invention. The system may also include at least some of the components for carrying out the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present invention may include a system for rapid detection of a microorganism of interest in a sample, comprising the above-mentioned device. For example, the device may include a first compartment comprising a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, the construct comprising a promoter and an indicator gene; the solid support comprising a binding component. In some embodiments, the system also comprises a handheld detection device.

[0201] The system according to the present technology or any of its components may be embodied in the form of a computer system, typical examples of which include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps constituting the methods of the present technology.

[0202] The computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include a memory. The memory may include a random access memory (RAM) and a read-only memory (ROM). The computer system may further include a storage device. The storage device may be a hard disk drive or a removable storage drive (floppy disk drive, optical disk drive, etc.). The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit connects the computer to other databases and the Internet via an I / O interface. The communication unit also transfers data to and receives data from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks (such as LANs, MANs, WANs, and the Internet). Thus, the computer system may facilitate input from a user via an input device accessible to the system via an I / O interface.

[0203] A computing device will typically include an operating system that provides executable program instructions for the general management and operation of the computing device, and will typically include a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) that stores instructions that, when executed by a processor of a server, cause the computing device to perform its intended functions. Suitable implementations of operating systems and general functions of computing devices are known or commercially available, and will be readily implemented by those of skill in the art, especially in light of the present disclosure.

[0204] To process input data, the computer system executes sequences of instructions stored in one or more memory elements, which may hold data or other information as needed. The memory elements may be in the form of information sources or physical memory elements present within the processing unit.

[0205] The environment can include the various data stores and other memory and storage media discussed above. These can be in various locations, such as local (and / or resident) to one or more of the computers, or on storage media remote from any or all of the computers via a network. In a particular set of embodiments, the information can be in a storage area network ("SAN") familiar to those skilled in the art. Similarly, any necessary files to perform functions attributed to a computer, server, or other network device can be stored locally and / or remotely, as necessary. Where the system includes computing devices, each such device can include hardware elements (e.g., elements including 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 (e.g., display device, printer, or speaker)) that can be electrically coupled by a bus. Such systems can also include one or more storage devices (such as disk drives, optical storage devices, and solid-state storage devices, such as random access memory ("RAM") or read-only memory ("ROM"), as well as removable media devices, memory cards, flash cards, and the like).

[0206] Such devices may also include the above-mentioned computer-readable storage medium reader, communication devices (e.g., modems, network cards (wireless or wired), infrared communication devices, etc.), and working memory. The computer-readable storage medium reader may be configured to connect to or receive computer-readable storage media, which may refer to remote, local, fixed, and / or replaceable storage devices, as well as storage media for containing, storing, transferring, and retrieving computer-readable information on a transient and / or more persistent basis. The systems and various devices will also typically include several software applications, modules, services, or other elements located within at least one working memory device, which may include an operating system and application programs (such as client applications or web browsers). It should be recognized that alternative embodiments may have many variations from the above. For example, customized hardware may also be used, and / or specific elements may be implemented in hardware, software (including highly portable software such as applets), or both. Additionally, they may be connected to other computing devices, such as network input / output devices.

[0207] Non-transitory storage media and computer readable media for containing code or portions of code may include any suitable media known or used in the art, including, but not limited to, storage media and communication media, including volatile and non-volatile, replaceable and non-replaceable media implemented in any method or technology for storing and / or transmitting information, such as computer readable instructions, data structures, program modules, or other data, including RAM, ROM, EEPROM, flash memory, or other storage technology, CD-ROM, digital versatile disk (DVD), or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage, or other magnetic storage, or any other medium that can be used to store the desired information and that can be accessed by a system device. Based on the disclosure and teachings provided herein, one of ordinary skill in the art will recognize other means and / or methods for implementing the various embodiments.

[0208] A computer-readable medium may include, but is not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, content addressable memories ("CAMs"), DDRs, flash memories (such as NAND flash or NOR flash), ASICs, configured processors, optical storage devices, magnetic tapes, or other magnetic storage devices, or any other medium from which a computer processor can read instructions. In one embodiment, a computing device may include a single type of computer-readable medium (such as random access memory (RAM)). In other embodiments, a computing device may include two or more types of computer-readable media (such as random access memory (RAM), disk drives, and caches). A computing device may communicate with one or more external computer-readable media (such as an external hard disk drive or an external DVD or Blu-ray drive).

[0209] As discussed above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronic devices (such as PLCs), programmable interrupt control circuits (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), or other similar devices.

[0210] The computing device includes a network interface. In some embodiments, the network interface is configured for communication over a wired or wireless communication link. For example, the network interface may be capable of communicating over a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, or the like. As another example, the network interface may be capable of communicating over a network such as CDMA, GSM, UMTS, or other cellular communication networks. In some embodiments, the network interface may be capable of point-to-point connection with another device via Universal Serial Bus (USB), 1394 FireWire, serial or parallel connections, or similar interfaces, or the like. Some embodiments of a suitable computing device may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include a data store in addition to or in lieu of a network interface.

[0211] Some embodiments of a suitable computing device may include or communicate with a number of external or internal devices, such as a mouse, a CD-ROM, a DVD, a keyboard, a display, audio speakers, one or more microphones, or any other input or output device. For example, a computing device may communicate with a variety of user interface devices and displays. Displays may use any suitable technology, including, but not limited to, LCD, LED, CRT, and the like.

[0212] The set of instructions for execution by a computer system may include various commands that instruct the processor to perform specific tasks, such as steps constituting the methods of the present technology. The set of instructions may be in the form of a software program. Furthermore, the software may be in the form of a collection of individual programs, program modules or portions of program modules with larger programs, such as the present technology. The software may also include modular programming in the form of object-oriented programming. Input data may be processed by the processor in response to user commands, results of previous processing, or requests made by another processor.

[0213] Although the present invention has been disclosed with reference to certain embodiments, many modifications, variations, 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, it is intended that the present invention not be limited to the described embodiments, but have full scope as defined by the language of the following claims and equivalents thereof. EXAMPLES

[0214] The following examples describe detection of small numbers of cells (even single bacteria) with reduced time to results, and are illustrative and not limiting of the invention. Example 1. Detection of a Microorganism of Interest in a Bacterial Culture

[0215] This example demonstrates the performance of the detection method using the device to successfully detect bacteria present in bacterial cultures. Assembly of the Apparatus

[0216] 100 μL A511 / P100 phage cocktail (1.2 × 10 9Pfu / mL) and 100 μL BHI medium + 1 mM CaCl2 were added to the top valve (first compartment) of the device. A511 / p100 phage is a phage that targets Listeria moncytogenes. A solid support press was used to press together with the top compartment of the device. The device tube was filled with 900 μL BHI medium + 1 mM CaCl2. The solid support was then placed in the device tube to absorb some of the medium. The assembled device (including the solid support immersed in the medium) was then kept at 4° C. overnight. Growth of bacterial cultures

[0217] Listeria moncytogenes was grown overnight in BHI medium (Becton Dickinson, Sparks, MD, USA) in a shaking incubator. The overnight culture was then passaged in BHI medium to log phase at 37°C. The log phase cells were then diluted to the appropriate cell number (see Figure 2). The solid support was removed from the device and the diluted culture was spiked onto the solid support at the indicated CFU levels. The solid support spiked with bacteria or medium only (control) was placed back into the device tube and the tube was gently shaken to mix the contents (Figure 2, Table 1).

[0218] In a similar experiment, two solid supports were spiked with 10 CFU of each bacteria. Non-seeded samples served as controls. These samples were incubated overnight at 35°C after infection and substrate exposure to allow bacterial numbers to expand (Figure 2, Table 2). infection

[0219] The snap valve was then broken by holding the solid support firmly and breaking the valve with thumb and index finger. Phage (200 μL of 6 × 10 8Pfu / mL) was expelled downwards by squeezing the valve on the top of the solid support tube. The contents in the solid support tube were mixed gently by swirling. The solid support was then placed in a 30° C. incubator for 4 hours to allow the phage to infect the bacteria. NanoGlo substrate (Promega, Madison, WI) was diluted 1:4 with 70% ethanol and 10 μL of the diluted substrate was placed in the solid support tube. The contents of the device tube were mixed by vortexing and then allowed to sit for 3 minutes. detection

[0220] Three detection methods were used: the solid support tube was inserted into a Hygiena handheld luminometer; 1 mL of the infection mixture was removed and transferred to a 1.5 mL microcentrifuge tube and read in a GloMax20 / 20 luminometer ("GloMax20 / 20"), and 150 μl of the infection mixture was transferred to a 96-well plate and read in a GloMax luminometer ("GloMax").

[0221] The solid supports were then soaked in medium overnight and detected at 2 hour infection times. The results are shown in Figure 2, Table 1, and Table 2, and graphical representations of the results are shown in Figures 3A and 3B. The results show that without growth enrichment (no overnight incubation of samples with solid supports prior to capture), the test was sensitive enough to detect 25,000 CFU with the Hygiena handheld luminometer (Figure 3A) (all readings of the bacterial seeded samples were above the baseline detection threshold of 10 relative luminescence units (RLU) for positive samples) and approximately 5,000 CFU with the GloMax20 / 20 or GloMax (Figure 3B). Example 2. Detection of Salmonella in Turkey Samples Assembly of the Apparatus

[0222] Add 100 μL SEA1 / TSP1 phage cocktail (1.2 × 10 7The device was assembled as described in Example 1, except that the tubes of the device were filled with 1 mL TSB medium and 100 μL TSB medium (ThermoFisher Oxoid, Grand Island, NY USA) and 1 mL SEA1 / TSP1 phage was a phage that targets Salmonella. Bacterial inoculation of ground turkey meat.

[0223] Salmonella cultures were grown overnight and diluted for high and low CFU samples as described below.

[0224] Test portions of 25g ground turkey were divided into three groups: uninoculated (5 samples), high inoculated (5 samples), and low inoculated (20 samples). Each 25g sample in the high inoculated group was inoculated with 2-10 CFU of Salmonella, and each sample in the low inoculated group was inoculated with 0.2-2 CFU of Salmonella. Samples were then placed into filtered sample bags and stored at 4°C for 48-72 hours. Bacterial enrichment in seeded ground turkey meat.

[0225] Pre-warmed TSB medium (41° C.) was added to each sample at a sample:medium ratio of 1:3. The samples were then blended on a STOMACHER® high for 30 seconds and then incubated at 41° C. without shaking for 24 hours to enrich for bacteria in the samples. Sampling

[0226] Test samples were obtained by immersing a solid support in each enrichment sample and swirling for 10 seconds to allow maximum sample absorption. The solid support was placed in a tube of the device filled with TSB medium. The tube was gently shaken to mix the contents within the tube and then either infected immediately or left at 37°C for an additional hour before infection. infection

[0227] The snap valve of the compartment containing the bacteriophage was then broken by holding the solid support firmly and breaking the valve with thumb and index finger. Phage (200 μL of 6 × 106 Pfu / mL) was expelled down the tube by squeezing the valve at the top of the device tube. The contents in the device tube were mixed gently by swirling. The solid support was then placed in a 37° C. incubator for 30 minutes or 2 hours to allow the phage to infect the bacteria. NanoGlo substrate (Promega, Madison, WI) was diluted 1:4 in 70% ethanol and 10 μL of the diluted substrate was placed in the device tube. The contents of the device tube were mixed by vortexing and then allowed to sit for 3 minutes. Signal was detected as described in Example 1. Results from the unseeded samples (control group) are shown in FIG. 4, Table 3, and results from the seeded samples (experimental group) are shown in Table 4. Graphical representations of the results are shown in FIG. 5A and FIG. 5B.

[0228] The results show that each of the three detection devices used was able to detect all turkey samples positive for Salmonella after 24 hours of culture enrichment. The signal from the GloMax and GloMax20 / 20 was much higher than the Hygiena luminometer. Incubation time (i.e., incubation of the solid support with medium that captured the bacteria prior to infection) and infection time are factors that can affect the signal strength. Of the various additional incubation and infection times tested, the sample with 0 hours incubation time and 2 hours infection time had the highest RLU, followed by the sample with 1 hour incubation time and 0.5 hours infection time, which in turn had the highest RLU, followed by the sample with 0 hours incubation time and 0.5 hours infection time. The results also show that the background signal for 0 hours incubation and 2 hours infection is the lowest. Example 3. Further studies to test the effect of infection time on the sensitivity of the assay

[0229] The experiment was set up as described in Example 2, except that after the solid support was submerged in the bacterial turkey sample, the solid support was placed in medium and infected immediately thereafter (i.e., there was no incubation time for the bacteria to grow on the solid support). Infection times ranged from 30 minutes to 2 hours. Signal was detected as described above. Results for three samples are shown in Figure 6, with the data plotted in Figures 7A-7C. The results show that, as shown in samples 24 and 26, the signal could be increased at 2 hours of infection, whereas in Hygiena, 30 minutes of infection showed no signal, but 2 hours of infection showed a signal.

[0230] 8A-8C show a comparison between the different devices GloMax and GloMax20 / 20. GloMax and GloMax20 / 20 showed similar results. Example 4. Testing of L. monocytogenes 19115 environmental sponge samples

[0231] L. monocytogenes was inoculated onto a ceramic tile surface, dried, and left at room temperature for 18-24 hours. A sample sponge was used to wipe the ceramic tile, and the sponge was placed in a bag and allowed to enrich at 35°C for 24 hours. The enrichment sample was then harvested using a solid support as described in Example 2. 100 μL Listeria phage (concentration 1.2 × 10 8 PFU / ml) was used to infect bacterial turkey cultures for 1 hour at 30° C. Signal was detected using GloMax and Hygiena. Results are shown in FIG. 9. The results show that the Hygiena handheld is capable of detecting L. moncytogenes contaminated environmental samples. Example 5. Different detection devices

[0232] The experiment was set up as described in Example 2. Phage infection time was 1 hour at 37° C. Signals were read on GloMax, 3M handheld luminometer ("3M"), and Hygiena, and the results are shown in FIG 10. It shows that 3M is more sensitive in signal detection.

Claims

1. A device, comprising: a first compartment comprising a recombinant bacteriophage having a genetic construct inserted into the bacteriophage genome, said construct comprising a promoter and an indicator gene, said indicator gene encoding an enzyme; and a second compartment comprising a signal generating component, said signal generating component facilitating detection of an indicator gene product produced following replication of the recombinant bacteriophage as a result of infection of the sample with said recombinant bacteriophage; wherein the device does not contain a culture medium.

2. The device of claim 1 , wherein the signal generating component is a substrate for the enzyme.

3. The device of claim 2 , wherein the enzyme is luciferase.

Images