Bacteriophage-based diagnostic test

Abstract

The present invention relates to a bacteriophage-based diagnostic test. In particular, the present invention relates to a diagnostic test for the detection of at least one target bacterium in a test sample, wherein the diagnostic test comprises at least one engineered bacteriophage specific to the or each target bacterium, wherein the or each bacteriophage comprises at least one signal molecule within or encapsulated in the head of the or each bacteriophage, and wherein the or each signal molecule is configured to be released from the head of the bacteriophage and produce a signal that can be detected upon the infection of the or each target bacterium with the or each bacteriophage, when the or each target bacterium is present in a test sample, in use.

Description

[0001] Bacteriophage-based Diagnostic Test

[0002] Technical Field of the Invention

[0003] The present invention relates to a bacteriophage-based diagnostic test. In particular, the present invention relates to a diagnostic test for the detection of at least one target bacterium, wherein the diagnostic test comprises at least one engineered bacteriophage specific to the or each target bacterium.

[0004] Background to the Invention

[0005] Bacteriophage (also known as phages) are viruses that specifically infect and replicate within host bacteria. Bacteriophage only infect specific species of host bacteria, and typically only infect a single bacterial species or a single bacterial strain. Bacteriophage do not infect non-bacterial cells (e.g. human cells). There are many types of bacteriophage having diversity in size, shape and genomic organisation. However, the majority of types of bacteriophage comprise a nucleic acid genome encapsulated within an icosahedral head of phage-encoded capsid proteins. The capsid proteins protect the bacteriophage genome and facilitate the delivery of the bacteriophage genome into the host bacteria upon infection. Bacteriophages have many applications in the biotechnology field, particularly in bacteriophage -based therapeutics or diagnostics. However, despite the increased research and development into such applications, there are very few bacteriophage-based diagnostic tests that are commercially available and used in clinics as standard tests.

[0006] The most common diagnostic methods for bacterial identification are culturebased tests, antibody (e.g. ELISA) tests, or nucleic acid amplification tests. Culture-based testing methods involve gene expression inside living bacteria. This results in diagnostic tests with limited diagnostic speed and that require a laboratory setting and specific reagents to perform the test. Tests involving antibodies (e.g. ELISA) may not be as accurate as other testing methods (such as DNA-based methods) due to the reduced sensitivity and specificity of antibody detection. Nucleic acid amplification tests require sample clean-up prior to testing and often involve complex methods steps that require a laboratory setting and the use of specific equipment to perform the test. Consequently, the main barriers of current bacterial diagnostic tests are that they are time-consuming, complex and have high associated costs.

[0007] Thus, it is an aim of embodiments of the present invention to provide a simple to use, low cost, and rapid diagnostic test for the detection of various bacterial strains. Further, embodiments of the present invention aim to provide a diagnostic method for the detection of bacterial strains that has the same or improved accuracy to known diagnostic methods. Additionally, embodiments of the present invention also aim to provide such a diagnostic test for use at home or in the field, wherein the test is simple to use and does not require high value equipment and reagents.

[0008] It is a further aim of embodiments of the present invention to provide a safe and effective engineered bacteriophage for use in a diagnostic test for the detection of various bacterial strains.

[0009] It is a further aim of embodiments of the present invention to overcome or mitigate at least one problem of the prior art, whether expressly described herein or not.

[0010] Summary of the Invention

[0011] According to a first aspect of the invention there is provided a diagnostic test for detecting the presence or the absence of at least one target bacterium in a test sample, the diagnostic test comprising at least one bacteriophage specific to the or each target bacterium, and wherein the or each bacteriophage comprises at least one signal molecule within or encapsulated in the head of the or each bacteriophage, and wherein the or each signal molecule is configured to be released from the head of the bacteriophage and thereby produce a signal that can be detected upon the infection of the or each target bacterium with the or each bacteriophage, when the or each target bacterium is present in a test sample, in use.

[0012] The signal produced by the or each signal molecule may only be detected when the or each target bacterium is present in the test sample. Thus, encapsulating the or each signal molecule inside the head of the or each bacteriophage harnesses the specificity of the interaction of a target bacterium with its specific (cognate) bacteriophage to allow for the specific detection of the target bacterium.

[0013] In some embodiments, when in use, the diagnostic test comprises a solution such as an aqueous medium. The solution may comprise the test sample and / or any buffer(s) and / or reagent(s) which may be added during use. In use, the solution may comprise one or more component(s) of the diagnostic test.

[0014] In some embodiments, the or each bacteriophage is an engineered bacteriophage. In some embodiments, the or each bacteriophage is a recombinant bacteriophage.

[0015] In some embodiments, the or each bacteriophage is a replication defective bacteriophage (i.e. does not produce progeny). The replication defective bacteriophage may have only loss of function compared to the wild type. Methods of producing types of replication defective bacteriophage are known (such as described in Lupo, D., et al.,

[0016] Virology, 2015, 486, 263-71) and any suitable type of replication defective bacteriophage may be used in the present invention. The use of replication defective bacteriophage is preferred in commercial applications as they are perceived to be safer in use.

[0017] In some embodiments, the diagnostic test may be specific to a given pathogen (i.e. a bacterial strain or a bacterial species that causes disease). In some embodiments, the diagnostic test may be specific to an illness (e.g. Urinary Tract Infections) involving more than one pathogen.

[0018] The diagnostic test may comprise one, two, three, four, five, six, seven, eight, or more than eight types of bacteriophage. A diagnostic test comprising more than one type of bacteriophage may provide the advantage of the diagnostic test being able to detect more than one type of target bacterium (i.e. different bacterial strains or species may be detected using one diagnostic test). In embodiments where the diagnostic test is specific to an illness involving more than one pathogen, the diagnostic test may comprise a bacteriophage specific to each causative bacterial strain or bacterial species. In such embodiments, each strain or species of bacteria may be detected in the same reaction on the device, or via specific reactions on the same device, or any combination in between.

[0019] In some embodiments, the or each bacteriophage is selected based on its specificity with the or each target bacterium. In some embodiments, the or each bacteriophage is independently selected from the group consisting of the following bacteriophage families: Podoviridae, Myoviridae, Siphoviridae, Microviridae, and any combination thereof.

[0020] In many cases, it is the tail of the bacteriophage that determines the specificity of the bacteriophage to a bacterial strain or bacterial species. Thus, in some embodiments, the or each bacteriophage may comprise a tail swap, wherein the tail of the or each bacteriophage is specific to the or each target bacterium. A tail swap describes when a tail of one bacteriophage is replaced with the tail of another bacteriophage, and wherein the tail swap does not affect the functioning of the bacteriophage. Bacteriophage tail swap methods are known in the field. The use of bacteriophages comprising tail swaps increases the number of potential bacterial targets available for detection.

[0021] In some embodiments, the or each target bacterium is independently selected from the group consisting of: bacteria that cause gonorrhoea (e.g. Neisseria gonorrhoeae), bacteria that cause chlamydia (e.g. Chlamydia trachomatis)' , bacteria associated with Bacterial Vaginosis (BV) (e.g. Gardnerella vaginalis, Prevotella, Peptostreptococcus, Bacteroides spp., Mobiluncus spp., Fusobacter spp., A. vaginae, and non-viridans group streptococci), bacteria that cause Urinary Tract Infections (UTIs) (e.g. uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Staphylococcus saprophyticus , Enterococcus faecalis, Enterococcus spp., group B Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus), group A Streptococcus (cause of bacterial sore throat also known as Strep Throat), bacteria that cause bacterial meningitis (e.g. most commonly Neisseria meningitidis, (meningococcus), Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, Streptococcus agalactiae (group B streptococcus), meningitis-associated Escherichia coli (MNEC)), other pathogenic strains of Escherichia coli (E. Coli) (e.g. enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), enteroinvasive Escherichia coli (EIEC) and diffusely adherent Escherichia coli (DAEC), uropathogenic Escherichia coli (UPEC), meningitis-associated Escherichia coli (MNEC), Escherichia coli pathotypes implicated in extraintestinal infections (ExPEC3)), Salmonella, Campylobacter, Listeria, and any combination of the aforementioned.

[0022] In preferred embodiments, the or each bacteriophage comprises one signal molecule encapsulated in or within the head of the or each bacteriophage.

[0023] The term “signal molecule” is used herein to describe a molecule or part thereof that is capable of producing a signal (otherwise known as acting as a reporter molecule), with such activity either on its own, or in combination with another molecule.

[0024] In some embodiments, the or each signal molecule is independently selected from the group consisting of: a colour-changing enzyme, a light-emitting enzyme, a part or parts of a light-emitting split-enzyme, a part or parts of a colour-changing split-enzyme, a small molecule dye, a nucleic acid intercalating dye, a substrate for a light-emitting or a colour-changing enzyme, and any combination thereof.

[0025] The or each signal molecule may be a protein, a peptide, or other expressiondependent, non-gene-based molecule.

[0026] At least one signal molecule may be capable of producing a light (e.g. luminescent, fluorescent, or bioluminescent) signal, or a colour signal (i.e. a specific colour). A light signal or a colour signal may only be detected upon release of the or each signal molecule from the head of the or each bacteriophage. For example, a signal molecule may produce a light signal which is not visible to the naked eye when it is within or encapsulated in the head of the or each bacteriophage but is visible when it is ejected from the head of the or each bacteriophage during or after infection of a bacterium by the or each bacteriophage. In some embodiments, a light signal or a colour signal may only be detected upon release of the or each signal molecule from the head of the or each bacteriophage and upon interaction with at least one molecule, reagent and / or substrate which effects production of a signal. The diagnostic test may therefore further comprise at least one further molecule, reagent and / or substrate which reacts with, interacts with, or binds with the or each signal molecule upon release of the or each signal molecule from the head of the or each bacteriophage, in use.

[0027] At least one signal molecule may be capable of producing a colour-change signal. A colour-change signal may be detected upon release of the or each signal molecule from the head of the or each bacteriophage, followed by binding of the signal molecule to at least one molecule, reagent and / or substrate which effects production of a signal, such as colour change, for example. The diagnostic test may therefore further comprise at least one further molecule, reagent and / or substrate which reacts with, interacts with, or binds with the or each signal molecule upon release of the or each signal molecule from the head of the or each bacteriophage, in use.

[0028] The diagnostic test may further comprise one or more agents capable of lysing (lysis agent) or opening the or each target bacterium, once the or each bacteriophage has infected the or each target bacterium. The or each signal molecule may be injected into the or each target bacterium, in use, and the one or more agents capable of lysing or opening the or each target bacterium then lyses or opens the bacterium to release or reveal the signal molecule(s).

[0029] The or each signal molecule may be injected into the target bacterium, in use, and the target bacterium lysed or opened to release the or each signal molecule. In some embodiments the diagnostic test comprises one or more substrates or molecules that react or bind with the or each signal molecule to produce the or each signal, only after the or each signal is released from the lysed or opened bacteria. In such embodiments, the or each substrate or molecule which reacts or binds with the or each signal molecule may not be injected into the or each target bacterium, such as being located in a medium in which the or each bacteriophage is present in the diagnostic test.

[0030] In some embodiments, the signal produced by the or each signal molecule comprises a colour-change of transparent to colour.

[0031] The or each signal molecule may comprise a coloured molecule which becomes visible on exit from the or each bacteriophage when the or each bacteriophage infects a target bacterium (or when the target bacterium is lysed or opened). For example, the or each signal molecule may comprise a red, yellow, green, purple or blue coloured molecule which colour is not visible to the naked eye when it is within or encapsulated in the or each head of the bacteriophage but is visible when it is ejected from the or each head of the bacteriophage during or after infection of a bacterium by the bacteriophage.

[0032] The or each signal molecule should be a suitable size for encapsulation inside the head of the or each bacteriophage. The or each signal molecule may fold (or form a tertiary structure) when inside the head of the or each bacteriophage. The or each signal molecule may un-fold in order exit the head of the or each bacteriophage through the bacteriophage capsid exit channel before or during ejection of the contents of the head of the bacteriophage into the target bacterium during infection. Once ejected, the or each signal molecule may re-fold to form in order to function and produce a signal.

[0033] The or each signal molecule (present in its functional structure) may have a diameter of less than 200 nm, less than 180 nm, less than 160 nm, less than 140 nm, less than 120 nm, less than 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, less than 20 nm, less than 10 nm, less than 8 nm, less than 6 nm, less than 4 nm, less than 2 nm, or less than 1 nm.

[0034] The or each signal molecule may have a molecular weight of less than 26 kDa, less than 24 kDa, less than 22 kDa, less than 20 kDa, less than 18 kDa, less than 16 kDa, less than 14 kDa, less than 12 kDa, less than 10 kDa, less than 9 kDa, less than 8 kDa, less than 7 kDa, less than 6 kDa, less than 5 kDa, less than 4 kDa, less than 3 kDa, less than 2 kDa, less than 1.8 kDa, less than 1.6 kDa, less than 1.4 kDa, less than 1.2 kDa, less than 1 kDa, less than 0.8 kDa, less than 0.6 kDa, less than 0.4 kDa, less than 0.2 kDa, or less than 0.1 kDa. Preferably, the or each signal molecule may have a molecule weight of between 0.1 kDa and 10 kDa, or more preferably between 0.1 kDa and 5 kDa.

[0035] The or each signal molecule may comprise less than 240, less than 220, less than 200, less than 180, less than 160, less than 140, less than 120, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20 amino acid residues. Preferably, the or each signal molecule may comprise between 50-200 amino acid residues, more preferably, between 60-100 amino acid residues.

[0036] In some embodiments, at least one signal molecule comprises one or more part of a split-enzyme (e.g. a light-emitting split-enzyme, a coloured split-enzyme, or a colourchanging split-enzyme). In such embodiments, one or more part of the split-enzyme may be encapsulated in or within the head of the or each bacteriophage, and the complementary part or parts of said split-enzyme are provided outside of the or each bacteriophage. The complementary part or parts of said split-enzyme may be provided and / or produced by the target bacterium. Alternatively, the complementary part or parts of said split-enzyme may be provided by another component of the diagnostic test (for example, when in use, the complementary part or parts of the split-enzyme may be free in a solution comprising part of the diagnostic test). Each part of the split-enzyme may not have any signalling activity alone, or at least any significant detectable amounts of signalling activity (e.g. each part alone does not emit a detectable about of light). In such embodiments, when the diagnostic test is in use, following the ejection of the one or more part the split-enzyme from the head of the or each bacteriophage and upon the binding of all parts of the split-enzyme, the re-united split-enzyme is capable of producing a signal. In some embodiments wherein the complementary part(s) of the splitenzyme are provided by another component of the diagnostic test, all of the parts of the split-enzyme may only be capable of binding once the target bacterium (containing the one or more part of the split-enzyme ejected from the head of the bacteriophage) is lysed. Lysis of the target bacterium may release the cellular contents of the target bacterium (including the ejected part(s) of the split-enzyme) so that the cellular contents are capable of interacting with or coming into contact with the other component or components of the diagnostic test which comprise or comprises the complementary part(s) of the splitenzyme. In such instances, the diagnostic test may further comprise a lysis agent (e.g. chemical or enzymatic agent) which causes the lysis of the target bacterium and the release of the cellular contents of the target bacterium.

[0037] The parts of the split-enzyme may have alpha-complementation.

[0038] In order for the split-enzyme to produce a signal, the parts of the split-enzyme may spontaneously re-unite when the diagnostic test is in use to form a functional enzyme, or alternatively, the parts of the split enzyme may be held together in close proximity by a protein tethering mechanism (e.g. SPYCATCHER-SPYTAG and

[0039] SnoopTag-SnoopCatcher, Hatlem. D., et al., Int. J. Mol. Sci., 2019, 30, 20(9)). In some embodiments, binding of the parts of the split-enzyme alone may not produce a signal and the interaction (e.g. binding) of at least one substrate and / or reagent is additionally required in order for the split-enzyme to produce the signal in use. Such substrate(s) and / or reagent(s) may be provided outside of the or each bacteriophage and via another component of the diagnostic test (for example, when the diagnostic test is in use, the reagent(s) and / or substrate(s) may be free in a solution comprising part of the diagnostic test).

[0040] In some embodiments, the or each split-enzyme comprises a smaller part and a larger counterpart. In such embodiments, the smaller part of the or each split-enzyme may be encapsulated in or within the head of the or each bacteriophage and the larger counterpart of the or each split-enzyme may be provided outside of the or each bacteriophage.

[0041] The use of a split-enzyme as a signal molecule has the advantage of allowing the use of signal molecules that may otherwise be too large to fit inside the head of the or each bacteriophage and / or are too large to be successfully injected into a bound target bacterium from the head of the bacteriophage through the bacteriophage capsid exit channel during infection of the target bacterium. The use of split-enzymes may also be useful for signal molecules which do not functionally re-fold following ejection from the head of the bacteriophage.

[0042] The or each split-enzyme may be selected from the group consisting of: Luciferases from Gaussia, Renuilla, Click beetle and Firefly; TEV Protease; Ubiquitin; Green fluorescent protein (GFP) and its derivatives ; P-lactamase; dihydrofolate reductase

[0043] (DHFR); P -galactosidase; Horseradish peroxidase (HRP); and any combination thereof. In some embodiments, at least one signal molecule is fused to at least one internal capsid protein of the or each bacteriophage. This may form a fusion protein (wherein the fusion protein comprises a signal molecule fused to an internal capsid protein). The term “internal capsid protein” is used herein to describe a capsid protein that is mostly or fully found on the inner surface of the capsid or is integrated and extends from the inner surface of the capsid or is partly or wholly occluded until the bacteriophage ejects its capsid contents.

[0044] In some embodiments, at least one signal molecule is fused to at least one leader peptide. The or each leader peptide is encoded from a leader sequence found in the genome of the or each bacteriophage. The or each leader peptide may localise the or each signal molecule within or encapsulates the signal molecule in the head of the or each bacteriophage.

[0045] In some embodiments, at least one signal molecule is a nucleic acid intercalating dye and intercalates with the bacteriophage genome encapsulated in or within the head of the or each bacteriophage.

[0046] In some embodiments, at least one signal molecule is encapsulated in or localised within the head of the or each bacteriophage during the formation of the or each bacteriophage. For example, at least one signal molecule may be localised within the head of the or each bacteriophage during the formation of the or each bacteriophage via a cell-free reaction (e.g. localised by diffusion).

[0047] In some embodiments, the diagnostic test further comprises at least one reagent and / or substrate required by the or each signal molecule to produce or enhance the signal.

[0048] The or each reagent and / or substrate may be provided outside of the or each bacteriophage. The or each reagent and / or substrate may be provided and / or produced by the target bacterium, or provided by another component of the diagnostic test (for example, when the diagnostic test is in use, the or each reagent or substrate may be free in a solution comprising part of the diagnostic test). When the diagnostic test is in use and when the or each signal molecule is released from the head of the or each bacteriophage, the or each reagent and / or substrate may contact and bind to the or each signal molecule to produce or enhance the signal. In embodiments wherein when in use the or each reagent and / or substrate is provided by another component of the diagnostic test, the or each reagent or substrate may only be capable of binding to the signal molecule once the target bacterium is lysed. Lysis of the target bacterium would release the cellular contents of the target bacterium (including the or each ejected signal molecule) so that the cellular contents are capable of interacting with the other component of the diagnostic test which comprises the or each reagent and / or substrate. In such instances, the diagnostic test may further comprise at least one lysis agent (e.g. chemical or enzymatic agent) which causes the lysis of the target bacterium and the release of the cellular contents of the target bacterium.

[0049] In a specific embodiment, at least one signal molecule is a part of nanoluciferase (NnLuc). It is known that nanoluciferase can be split into an N-terminal fragment and a C-terminal fragment to result in split-nanoluciferase (Nelson, T. J. et al., Methods Enzymol., 2019, 622, 55-66). Nanoluciferase may be split into a N-terminal fragment comprising amino acid residues 1-65, and a C-terminal fragment comprising amino acid residues 66-171. In some embodiments, the N-terminal fragment of split-nanoluciferase (NnLuc) (i.e. the smaller fragment) is encapsulated in or within the head of the or each bacteriophage and the C-terminal fragment of split- nanoluciferase (CnLuc) (i.e. the larger fragment) is provided outside of the or each bacteriophage. The NnLuc may form part of a fusion protein with an internal capsid protein. When the diagnostic test is in use, when the N-terminal fragment of split-nanoluciferase is ejected from the head of the or each bacteriophage during infection, the two fragments of split-nanoluciferase may bind and regain the ability to emit light upon the further binding of a substrate, such as furimazine. In such embodiments, the diagnostic test may further comprise the substrate furimazine which may be provided outside of the or each bacteriophage. The re-united split- nanoluciferase may only emit light in the presence of the substrate furimazine or an equivalent, compatible substrate.

[0050] In a specific embodiment, the diagnostic test comprises a bacteriophage from the T7 family of bacteriophages for the detection of Escherichia coli (E. coh). Even more specifically, the diagnostic test may comprise a K1F bacteriophage (a T7-like bacteriophage) for the detection of E. coli EV36 strain. The bacteriophage may comprise a fusion protein comprising an internal capsid protein and a signal molecule. The fusion protein may comprise an internal capsid protein selected from a group consisting of: gp6.7, gp7.3, gpl4, gpl5, and gpl6. Preferably, the fusion protein comprises the internal capsid protein gp6.7 or gpl4. The fusion protein may additionally comprise the N- terminal fragment of split- nanoluciferase (NnLuc). In such embodiment, the C-terminal fragment of split-nanoluciferase (CnLuc) and the substrate furimazine are provided outside of the bacteriophage via a solution comprising part of the diagnostic test when in use.

[0051] In some embodiments, the diagnostic test may be provided on a test strip. The diagnostic test may further comprise a test strip, wherein the test strip comprises the or each bacteriophage. The test strip may also comprise a part or parts of a split-enzyme and / or at least one reagent and / or substrate required by the or each signal molecule to produce or enhance the signal.

[0052] Prior to use of the diagnostic test, the or each bacteriophage, signal molecule, and / or reagent and / or substrate may be provided on the test strip as a solid, solid particulate and / or powder. Proteins and enzymes may be provided as lyophilised or otherwise dried solids, solid particulates and / or powders on the test strip, prior to use. In use, the user may apply a liquid to the test strip in order to initiate the test and to form a solution for use. The liquid applied may be in the form of a test sample and / or a buffer solution. In use, the or each bacteriophage, signal molecule, and / or reagent and / or substrate may contact the solution in use and solubilise in the solution.

[0053] The test strip may comprise a polymer-based strip comprising the components of the diagnostic test. The components of the diagnostic test may be dried onto the polymer- based test strip.

[0054] The test strip may be a lateral-flow test strip, or a static assay strip.

[0055] Any suitable known and / or commercially available test strip may be used for the diagnostic test of the first aspect of the invention. Examples of suitable commercially available test strips include: Abacm Universal Lateral Flow Assay Kit, ab270537.htm]; Milenia Biotec HybriDetect - Universal Lateral Flow Assay Kit, DDTD Universal lateral flow assay, h ttps : / / ww w.ddtd.org / muversal-1 ateral -flow-assay . The use of such test strips are generally known and therefore would be familiar and simple to perform by the majority users.

[0056] In some embodiments, the test strip may comprise an area for application of the test sample and / or the buffer solution by the user.

[0057] In some embodiments, readout may comprise one locus on the test strip, or more than one locus. In some embodiments, one locus on the test strip provides a readout for the detection of any one of the target bacterium present in the test sample. In some embodiments wherein there is more than one locus on the test strip, each locus may provide a readout for the detection of one target bacterium.

[0058] In some embodiments, the test strip may comprise at least one control signal that indicates whether the test has been performed correctly.

[0059] The test strip may indicate the presence of at least one target bacterium (i.e. a positive result) by providing a signal, for example, the appearance of a colour on the test strip. The test strip may indicate the absence of at least one target bacterium (i.e. a negative result) by the absence of a signal (e.g. colour) on the test strip. Alternatively, a negative result may be indicated by the appearance of a signal on the test strip, wherein the signal is different to the signal provided when a positive result is obtained. For example, a negative result may result in the appearance of a colour (e.g. blue) on the test strip, wherein the colour is different to the colour that appears when a positive result (e.g. red) is obtained. A colour signal may be accompanied by a symbol indicator for additional clarity; for example, a positive and / or negative colour signal may be accompanied by a positive (+) and negative (-) sign, respectively. The diagnostic test may produce a rapid result. The diagnostic test may detect the presence or the absence of the or each target bacterium in a test sample in less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 45 seconds, less than 30 seconds, or less than 15 seconds. Preferably, the diagnostic test may detect the presence or the absence of the or each target bacterium in a test sample in less than 7 minutes, more preferably less than 5 minutes, or even more preferably around 1 minute or less. Thus, the diagnostic test provides a rapid result and is quicker than other standard diagnostic testing methods (e.g. DNA-based diagnostic tests).

[0060] According to a second aspect of the invention, there is provided a diagnostic test according to the first aspect of the invention for use in medical testing. The diagnostic test may detect the presence or the absence of at least one bacterial infection in a test sample. The test sample may comprise a biological fluid, taken from a subject. The biological fluid may be selected from the group consisting of: blood, serum, plasma, urine, faeces, interstitial fluid, phlegm, sputum, mucous, semen, vaginal mucosa, skin and cerebral fluid. The diagnostic test may detect the presence or the absence of at least one bacterial infection independently selected from the group consisting of: gonorrhoea, chlamydia, bacterial vaginosis, urinary tract infections, strep throat, bacterial meningitis, and any combination thereof.

[0061] According to a third aspect of the invention, there is provided the use of a diagnostic test according to the first aspect of the invention in food or beverage testing. The diagnostic test may detect the presence or the absence of bacterial contamination in a food or beverage product. The food product may be in the form a liquid, a suspended solid, a gel, or a paste, for example. The food product may comprise a food item suspended in an aqueous medium or comprising a liquid or paste formed from a fluidised or homogenised solid, for example. The food product may have been agitated prior to performing the diagnostic test for the purpose of transferring any putative bacteria into the aqueous medium. The food product may be selected from the group consisting of: meat or meat-based food products, cereals, confectionery, ice-cream, frozen food products, cheese, baked food products, fruit, and vegetables. The beverage may be selected from the group consisting of: milk or milk based beverages, tea, coffee, alcoholic beverages, soda, fruit juice, carbonated beverages and smoothies. The diagnostic test may detect the presence or absence of at least one bacterium from the bacterial species, strain, serotype, serogroup or serovar independently selected from the group consisting of: pathogenic food-bome E. Coli strains, subtypes, serovars, serotypes and / or serogroups such as but not limited to enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC) meningitis-associated E. coli (MNEC) and non-0157 Shiga toxin-producing E.coli (STEC) (the so-called “big six” serogroups that cause most non-0157 infections: 026, 045, 0103, 0111, 0121, and 0145 serogroups); Salmonella', Campylobacter, Listeria monocytogenes', Staphylococcus aureus', and any combination of the aforementioned.

[0062] According to a fourth aspect of the invention, there is provided the use of a diagnostic test according to the first aspect of the invention in water testing. The diagnostic test may detect the presence or the absence of bacterial contamination in a water sample. The water sample may comprise a sample selected from the group consisting of: waste-water (such as domestic, industrial or commercial waste-water, for example), sewage, drinking water, coolant, river water, lake water and sea or ocean water. The diagnostic test may detect the presence or the absence of at least one bacterium independently selected from the group consisting of: E. Coli, Salmonella spp. , Shigella spp., Legionella pneumophila, Campylobacter spp., general coliforms, and any combination thereof.

[0063] According to a fifth aspect of the invention, there is provided method of detecting at least one bacterium, the method comprising:

[0064] (i) providing a diagnostic test comprising at least one bacteriophage specific to at least one bacterium, and wherein the or each bacteriophage comprises at least one signal molecule within or encapsulated in the head of the or each bacteriophage, and wherein the or each signal molecule is configured to be released from the head of the bacteriophage and thereby produce a signal that can be detected upon the infection of at least one target bacterium with the or each bacteriophage , when the or each target bacterium is present in a test sample, in use;

[0065] (ii) contacting the or each bacteriophage with a test sample;

[0066] (iii) incubating the at least one bacteriophage with the test sample; and

[0067] (iv) detecting the presence or the absence of a signal from the at least one signal molecule, wherein the presence of a signal indicates the presence of at least one bacterium specific to the or each bacteriophage.

[0068] In some embodiments, the diagnostic test is a diagnostic test according to the first aspect of the invention. In some embodiments, step (i) further comprises providing at least one reagent and / or substrate required by the or each signal molecule to produce a signal in the presence of the or each bacterium. The at least one reagent and / or substrate may be as described for the first aspect of the invention. The at least one reagent and / or substrate may comprise part of the diagnostic test, or the at least one reagent and / or substrate may be added to the diagnostic test in use.

[0069] In some embodiments, the or each signal produces a light and / or colour signal and step (iv) comprises detecting said light and / or colour signal. Alternatively, or additionally, the or each signal generates a colour-change and step (iv) comprises detecting said colour-change. The or each signal molecule may comprise a coloured molecule which becomes visible on exit from the head of the or each bacteriophage when the or each bacteriophage infects a bacterium in use.

[0070] In some embodiments, the at least one bacteriophage is incubated with the test sample for a time period of less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 45 seconds, less than 30 seconds, or less than 15 seconds. Preferably, the or at least one bacteriophage is incubated with the test sample for a time period of less than 7 minutes, more preferably less than 5 minutes, or even more preferably around 1 minute.

[0071] Following incubation (step (iii)), the signal may be immediately detected.

[0072] According to a sixth aspect of the invention, there is provided a method of encapsulating a signal molecule inside the head of a bacteriophage, the method comprising engineering the bacteriophage with at least one fusion gene, wherein the or each fusion gene comprises a gene encoding an internal capsid protein of the bacteriophage genome and a gene encoding a signal molecule.

[0073] The bacteriophage, the internal capsid protein, the signal molecule, or the components thereof may be as described for the first aspect of the invention.

[0074] In some embodiments, the method comprises directly engineering the bacteriophage genome to incorporate the or each fusion gene. In such embodiments, a gene encoding an internal capsid protein is selected from the bacteriophage genome and the bacteriophage genome is directly engineered by fusing a gene encoding a signal molecule with the gene encoding the selected internal capsid protein to form the or each fusion gene. Providing the fusion gene directly to the bacteriophage genome may be readily done using standard CRISPR / Cas selection methods.

[0075] In some embodiments, the method comprises removing from the bacteriophage genome, or supressing, the gene encoding the internal capsid protein (this may be readily done using standard CRISPR / Cas selection methods) and providing said gene to the bacteriophage genome as part of the or each fusion gene via the use of at least one vector. In some embodiments, the use of a vector transforms the fusion gene into the genome of the or each bacteriophage. In some embodiments the vector comprises the fusion gene, comprising the signal molecule fused to a bacteriophage capsid-localising protein (internal capsid protein) or a leader sequence. Transformation of the or each target bacterium with the expression of vector-bome fusion protein prior to or coincident with infection by the cognate (specific) bacteriophage results in non-genomically-engineered progeny bacteriophage harbouring the fusion protein but composed of a wild-type genome. In one embodiment, the bacteriophage comprises a deleted gene which prevents replication of the bacteriophage, until a fusion gene which comprises the deleted gene and the gene encoding the signal molecule is supplied in trans, possibly via a plasmid vector or cell free expression. Progeny bacteriophage thus produced are capable of infecting target bacteria and releasing the signal molecule, but cannot themselves produce replication-competent progeny. The bacteriophage may therefore be replication defective (lacking a gene which enables replication) and may only function to create viable progeny when the vector provides the deleted gene, in situ, in the bacterium in which the bacteriophage is being propagated.

[0076] In some embodiments, the or each vector may be a plasmid.

[0077] In some embodiments, the fusion gene and / or vector may also comprise at least one promotor. The or each promotor may amplify the expression of the gene encoding the internal capsid protein and / or the signal molecule.

[0078] In some embodiments, the fusion gene may be split, and parts thereof may be supplied on separate vectors. One or more of the vectors may be supplied as a non- encapsidated helper plasmid. This may occur if larger fusion genes are used.

[0079] According to a seventh aspect of the invention, there is provided a method of encapsulating a signal molecule inside the head of a bacteriophage via a cell-free expression system, the method comprising contacting a cell-free preparation with the genome of a bacteriophage and at least one fusion gene in a cell-free expression reaction, wherein the or each fusion gene comprises a gene encoding an internal capsid protein and a gene encoding a signal molecule. In some embodiments, the cell-free expression reaction yields at least one bacteriophage comprising at least one signal molecule within or encapsulated in the head of the or each bacteriophage.

[0080] Cell-free expression systems and methods are known. Cell-free expression systems are also known as TXTL (transcription translation) expression systems. Any suitable known systems or methods may be used for the seventh aspect of the present invention.

[0081] In some embodiments, the cell-free expression reaction comprises a single reaction mixture comprising all enzymes and reagents required to express proteins based on any added gene sequence.

[0082] The bacteriophage, the internal capsid protein, the signal molecule, or the components thereof may be as described for the first aspect of the invention.

[0083] In some embodiments, the genome of the bacteriophage may be modified before adding the genome to the cell-free expression reaction. In one embodiment, the modification may comprise deleting the gene that encodes the internal capsid protein (this may be done using standard CRISPR / Cas selection methods).

[0084] In some embodiments, the genome of the or each bacteriophage formed by the cell-free reaction contains the fusion gene.

[0085] An internal capsid protein describes a capsid protein that is mostly or fully found on the inner surface of the capsid or is integrated and extends from the inner surface of the capsid or is partly or wholly occluded until the bacteriophage ejects its capsid contents. In some embodiments, the or each fusion gene is supplied to the cell-free expression reaction via at least one vector.

[0086] In some embodiments, the or each vector may be a plasmid.

[0087] In some embodiments, the or each fusion gene may also comprise at least one promotor to amplify the expression of the internal capsid protein and / or the signal molecule.

[0088] In some embodiments, the or each fusion gene may be split, and parts thereof may be supplied on separate vectors.

[0089] According to further aspects of the invention, there are provided methods of encapsulating a signal molecule inside the head of a bacteriophage according to the sixth or seventh aspects of the invention, but wherein the gene encoding an internal capsid protein is replaced by a leader peptide sequence in the fusion gene. A leader peptide sequence may direct the gene encoding the signal molecule during bacteriophage formation so that the signal molecule is encapsulated in the head of the bacteriophage.

[0090] According to an eighth aspect of the invention there is provided a method of encapsulating a small molecule inside the head of a bacteriophage via a cell-free expression system, the method comprising contacting a cell-free preparation with the genome of a bacteriophage in a cell-free expression reaction and providing a small molecule to the cell-free expression reaction, and wherein the small molecule is a signal molecule or a substrate molecule for a signal molecule.

[0091] In some embodiments, the cell-free expression reaction yields at least one bacteriophage comprising at least one small molecule within or encapsulated in the head of the or each bacteriophage. Cell-free expression systems and methods are known. Cell-free expression systems are also known as TXTL (transcription translation) expression systems. Any such known systems / methods may be used for the eighth aspect of the present invention.

[0092] In some embodiments, the cell-free expression reaction comprises a single reaction mixture comprising all enzymes and reagents required to express proteins based on any added gene sequence.

[0093] The bacteriophage, the internal capsid protein, the signal molecule, or the components thereof may be as described for the first aspect of the invention.

[0094] In some embodiments, the small molecule may be a small molecule dye. During the cell-free expression reaction and bacteriophage synthesis, some of the small molecule dye may be localised inside the head of the bacteriophage by diffusion.

[0095] In some embodiments, the small molecule may be a DNA intercalating dye. During the cell-free expression reaction and bacteriophage synthesis, the DNA intercalating dye may have affinity for the bacteriophage genomic DNA and thus when the bacteriophage DNA is being packaged inside the head of the bacteriophage, the DNA intercalating dye will also be encapsulated inside the head of the bacteriophage.

[0096] In other embodiments, the small molecule may be a small molecule reagent or substrate required by a larger signal molecule (e.g. a light-emitting enzyme) to generate a signal. For example, the small molecule may be furimazine, which is required by nanoluciferase to generate a signal, or any other molecule that can be used by nanoluciferase to generate a signal.

[0097] The small molecule must be a suitable size for encapsulation inside the head of the or each bacteriophage. The small molecule may be suitable for encapsulation inside the head of a bacteriophage having a diameter of less than 200 nm, less than 180 nm, less than 160 nm, less than 140 nm, less than 120 nm, less than 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, or less than 20 nm.

[0098] The small molecule may have a molecular weight of less than 1800 g mol1, less than 1600 g mol1, 1200 g mol1, less than 1000 g mol1, less than 900 g mol1, less than 800 g mol1, less than 700 g mol1, less than 600 g mol1, less than 500 g mol1, less than 400 g mol1, less than 300 g mol1, less than 200 g mol1, or less than 100 g mol1. Preferably, the small molecule may have a molecular weight of between 100 g mol1- 1400 g mol1, or more preferably, 100 g mol1- 1000 g mol1.

[0099] The small molecule may be selected from the group consisting of: Coomassie Brilliant Blue, Ponceau S, Amido Black 10B, Bicinchoninic Acid, Ninhydrin, Sudan Black, ethidium bromide, SYBR Green, Propidium Iodide, Acridine Orange, DAPI, Hoechst 33258, Hoechst 33342, 7-Aminoactinomycin D (7-AAD), TOTO-1, and YOYO- 1.

[0100] A bacteriophage formed by the methods of the sixth, seventh and eighth aspects of the present invention may be used for the diagnostic test of the first to fourth aspects of the invention.

[0101] Detailed Description of the Invention

[0102] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0103] Figure 1 illustrates the mechanism of an embodiment of the diagnostic test according to the invention, in use, (A) wherein the presence of a target bacterium in a test sample is detected and (B) when the test sample is absent of a target bacterium.

[0104] Figure 2 shows the activity of signal molecules (NnLuc) released from the heads of engineered bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc), thus demonstrating the use of these components in an embodiment of the diagnostic test of the present invention. KlFe6.7:NnLuc (A) and KlFel4:NnLuc (B) were heat-treated at the indicated temperatures for 5 min, and mixed with CnLuc extracted from producer cells. The mean values of three parallel luminescence measurements are shown. Asterisks mark the results of two-tailed, unpaired t tests comparing each value to the background signal (LB). *: p<0.01; **: p<0.0001

[0105] Figure 3 shows the activity of signal molecules (NnLuc) released from the heads of engineered bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc) similarly to Figure 2 but compared to control samples. BL21CnLuc marks extracts of E. coli BL21(DE3) cells harbouring induced pZA31CnLuc_his_tetR plasmid, BL21 marks plasmidless E. coli BL21(DE3) cells, KlFe6.7 marks KlFe6.7:NnLuc bacteriophages and KlFel4 marks KlFel4:NnLuc bacteriophages. The mean values of three parallel luminescence measurements are shown. Asterisks mark the results of two-tailed, unpaired t tests comparing each value to the control (BL21(DE3) + KlFwt) signal. *: p<0.05

[0106] Figure 4 shows the endpoint luminescence signals produced by embodiments of the diagnostic test of the invention and indicating various bacteriophage- bacterial interactions. EV36CnLuc marks E. coli EV36 cells harbouring induced pZA3 lCnLuc_his_tetR plasmid, EV36 marks plasmidless E. coli EV36 cells, NissleCnLuc marks E. coli Nissle 1917 cells harbouring induced pZA31CnLuc_his_tetR plasmid. E. coli EV36 + induced pZA31CnLuc_his_tetR (EV36CnLuc) or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (NissleCnLuc) were challenged with KlFe6.7:NnLuc or KlFel4:NnLuc bacteriophages. The mean values of three parallel luminescence measurements are shown. Asterisks mark the results of two-tailed, unpaired t tests comparing each value to the negative control (plasmidless E. coli EV36 + K1F wt phage). *: p<0.001

[0107] Figure 5 shows the kinetics of the luminescence signal produced by embodiments of the diagnostic test of the invention shown in Figure 3. E. coli EV36 + induced pZA31CnLuc_his_tetR (EV36CnLuc) or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (NissleCnLuc) were challenged with KlFe6.7:NnLuc or KlFel4:NnLuc bacteriophages. The reaction was stopped by the addition of the Nano-Gio® Luciferase Assay at the time indicated, and the luminescence was recorded. The mean values of three parallel measurements are shown. Asterisks mark the results of two- tailed, unpaired t tests comparing each value to the negative control (plasmidless E. coli EV36 + the respective phage). *: p<0.01

[0108] Figure 6 shows the kinetics of the luminescence signal produced by embodiments of the diagnostic test of the invention wherein E. coli EV36 + induced pZA31CnLuc_his_tetR (EV36CnLuc) or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (NissleCnLuc) were challenged with KlFe6.7:NnLuc or KlFel4:NnLuc bacteriophages. The mean values of three parallel measurements are shown. Asterisks mark the results of two- tailed, unpaired t tests comparing each value to the negative control (plasmidless E. coli EV36 + the respective phage). *: p<0.01

[0109] Figure 7 shows a graph of the luminescence signal detected at 5 minutes 45 seconds after E. coli EV36 + induced pZA31CnLuc_his_tetR (EV36CnLuc) or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (NissleCnLuc) were challenged with KlFe6.7:NnLuc (A) or KlFel4:NnLuc (B) bacteriophages. The mean values of three parallel measurements are shown. Asterisks indicate the results of two-tailed, unpaired t tests comparing the values obtained with the two bacterial strain to each other. *: p<0.05; **: p<0.001.

[0110] Figure 8 shows the luminescence detected at various time points after E. coli EV36

[0111] + induced pZA3 lCnLuc_his_tetR (EV36CnLuc) or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (NissleCnLuc) were challenged with KlFe6.7:NnLuc (A) or KlFel4:NnLuc (B) bacteriophages. Rifampicin was added at 4 minutes (arrow) to E. coli EV36 + induced pZA31CnLuc_his_tetR (EV36CnLuc + rifampicin). The mean values of three parallel measurements are shown. Asterisks indicate the results of two-tailed, unpaired t tests comparing the values obtained with E. coli Nissle 1917. *: p<0.005; **: p<0.001.

[0112] Figure 9 illustrates a diagnostic test comprising a test strip according to an embodiment of the present invention, wherein (A) shows a test strip that shows only a positive signal and (B) shows a test strip that shows both a positive and negative signal, and wherein “C” is the control signal and “S” is the sample signal.

[0113] The following example describes embodiments of the diagnostic test of the invention, wherein the diagnostic test (Figure 1) comprises an engineered K1F bacteriophage 1 (a T7-like bacteriophage) for the detection of the presence (Figure 1 A) or absence (Figure IB) of E. coli EV36 strain protected by a KI capsule (i.e. the cognate host of a K1F bacteriophage) by detecting the presence 2 or absence 3 of a luminescence signal, respectively. The diagnostic test comprises a split-nanoluciferase protein as the signalling molecule, wherein the split-nanoluciferase comprises a C-terminal fragment and an N-terminal fragment (Nelson, T. J., et al., Methods Enzymol., 2019, 622, 55-66). These enzyme fragments spontaneously bind to each other in solution and thereby regain the ability to emit light in the presence of the substrate compound furimazine. The N- terminal fragment 4 is encapsulated in the head of the bacteriophage 5 by forming a fusion protein with an internal capsid protein of the bacteriophage genome 6. In one embodiment, the N-terminal fragment of split-nanoluciferase is fused to the internal capsid protein, gp6.7, of a KI F bacteriophage. In another embodiment, the N-terminal fragment of split nano-luciferase is fused to the internal capsid protein, gpl4, of a K1F bacteriophage. The C-terminal fragment 7 of split-nanoluciferase is then provided outside of the engineered K1F bacteriophage 1 free in solution, along with furimazine 8 (provided via the Nano-Gio® Luciferase Assay).

[0114] Figure 1 illustrates the mechanism of the embodiments of the diagnostic test summarised above in use. Upon binding of the E. coli EV36 cell 9 to the engineered K1F bacteriophage 1 of either embodiment above, the fusion protein (i.e. the N-terminal fragment of split- nanoluciferase fused with gp6.7 or gpl4), is injected into the bound E. coli EV36 cell 9. Following infection of the E. coli EV36 cell 9 by the engineered K1F bacteriophage 1, the bacterium cell is lysed 10 (using Nano-Gio® Luciferase Buffer) releasing the bacteriophage DNA 6 and N-terminal fragment of split-nanoluciferase 4 into solution. The N-terminal fragment of split-nanoluciferase 4 is now free to interact with the C-terminal fragment of split nano-luciferase 7 and furimazine 8 that are free in solution. The presence of E. coli EV36 can be detected due to an arising luminescence signal generated by the binding of the re-united split-nanoluciferase 11 with furimazine 8.

[0115] Bacteriophage engineering

[0116] Two types of recombinant K1F bacteriophages, KlFe6.7NnLuc and KlFel4NnLuc, were engineered for use in the two embodiments of the diagnostic test of the present invention described above. The method of engineering the recombinant K1F bacteriophages described herein is in accordance with an embodiment of a method of encapsulating a signal molecule within the head of the bacteriophage.

[0117] Recombinant bacteriophage KlFe6.7NnLuc carries an extra copy of gene g6.7 (SEQ ID NO: 1) in translational fusion with the gene sequence encoding the N-terminal fragment of split- nanoluciferase (NnLuc) (g6.7:NnLuc, SEQ ID NO: 2), and wherein said fusion gene is integrated into the bacteriophage genome downstream of gene glOb (the minor capsid protein of K1F). Recombinant bacteriophage KlFel4 NnLuc carries an extra copy of gene gl4 (SEQ ID NO: 3) in translational fusion with the gene sequence encoding NnLuc (gl4:NnLuc, SEQ ID NO: 4), and wherein said fusion gene is integrated into the bacteriophage genome downstream of gene glOb. For the production of the KlFe6.7NnLuc and KlFel4NnLuc recombinant bacteriophages, two donor plasmids were firstly constructed, pSBC3g6.7:NnLuc (SEQ ID NO: 5) and pSBC3gl4:NnLuc (SEQ ID NO: 6), respectively. A donor plasmid (pSBC3, sequence published; Mpller-Olsen, C., et al., Sci. Rep., 2018, 8, 17559) was used as basis, and the gene sequence encoding NnLuc was integrated with gene g6.7 or gl4 (including the promoters of each respective gene) as a fusion gene. The gene sequence encoding NnLuc was designed with a flexible linker at its N terminus (SEQ ID NO: 7 for DNA).

[0118] To generate the pSBC3g6.7:NnLuc (SEQ ID NO: 5) donor plasmid, PCR amplification of the pSBC3 donor plasmid, the gene sequence encoding NnLuc (SEQ ID NO: 7), and gene g6.7(including the promoter of gene g6.7) from K1F bacteriophage lysate resulted in three PCR products that were assembled in a Circular Polymerase Extension Cloning (CPEC) reaction (Quan, J., et al., PloS One, 2009, 4, e6441). The resulting product was then purified and electroporated into E. coli MDS42, followed by plating on LB + Ampicillin agar plates to result in plasmids pSBC3g6.7:NnLuc (SEQ ID NO: 5).

[0119] To generate the pSBC3gl4:NnLuc donor plasmid (SEQ ID NO: 6), the above procedure was followed but instead involving the PCR amplification of gene gl4 (including the promoter of gene gl4), rather than g6.7.

[0120] Subsequently, to construct the KlFe6.7NnLuc recombinant bacteriophage, the donor plasmid pSBC3g6.7:NnLuc was transformed into E. coli EV36. To do so, K1F bacteriophage was grown on E. Coli EV36 harbouring the donor plasmid pSBC3g6.7:NnLuc. The resulting bacteriophage mix was grown on E. Coli EV36 + pCas9KlFC2 (obtained from Molecular Cloud: https: / / w ww.n^o]eculardoud.org / pIasmid / pCas9 10 / MC-0000792.html) for three rounds of phage growth in order to select for recombinant K1F bacteriophage over the wild type K1F bacteriophage. The third phage lysate was diluted to obtain isolated plaques on a lawn of E. coli EV36, and the plaques were screened by plaque PCR.

[0121] To construct the KlFel4NnLuc bacteriophage, the above procedure was followed but the donor plasmid pSBC3gl4:NnLuc was instead used.

[0122] Cloning of the gene encoding CnLuc for bacterial expression

[0123] In order to test the embodiments of the diagnostic test of the invention, CnLuc must be provided outside of the bacteriophage. In the example herein, CnLuc is expressed by the target bacterial strain so that upon release of the NnLuc from the head of the bacteriophage into the bound bacterial cell on infection, the enzyme parts can reunite and bind and regain the capability to produce a signal. However, this mechanism is provided as proof of concept only and preferably the CnLuc is provided free in solution when the diagnostic test is in use.

[0124] In the example herein, the gene sequence encoding CnLuc (SEQ ID NO: 8) was cloned for bacterial expression. To do so, the gene sequence encoding CnLuc (SEQ ID NO: 8) was translated into the genome of target bacterial cells for bacterial expression. The gene sequence encoding CnLuc was translated into bacterial cells using a plasmid, named pZA31CnLuc_his_tetR herein, but any suitable plasmid will suffice.

[0125] Sample Preparation for Luminescence Tests 1-4

[0126] Samples were prepared for luminescence analysis. Nano-Gio® Luciferase Assay

[0127] Substrate and Buffer was added to the various test samples described in Tests 1 - 4 below, prior to instantly recording the luminescence of each sample. Nano-Gio® Luciferase Assay Substrate contains furimazine which is the substrate required to generate the luminescence signal when NnLuc binds CnLuc. Nano-Gio® Luciferase Assay Buffer contains a lysis reagent which allows for furimazine and CnLuc to re-unite and bind with NnLuc.

[0128] Test 1 (In Vitro) - Release of the fusion proteins from the bacteriophage head

[0129] Test 1 demonstrates that the fusion genes (g6.7:NnLuc and gl4:NnLuc) of the recombinant K1F bacteriophages (KlFe6.7NnLuc and KlFel4NnLuc) for use in the embodiments of the diagnostic test of the invention are successfully packaged into the bacteriophage heads. Test 1 involves the heat inactivation (at 70 °C, 75 °C and 85 °C) of each recombinant K1F bacteriophages (KlFe6.7NnLuc and KlFel4NnLuc) so that all capsid-enclosed proteins are released.

[0130] In order to detect the release of the fusion proteins from each recombinant K1F bacteriophage head, a culture of E. Coli BL21(DE3) harbouring a plasmid comprising the gene sequence encoding CnLuc (pZA31CnLuc_his_tetR) was grown in liquid LB medium + aTc. Cells were pelleted at 10.000 g for 1 minute, and resuspended in 500 pl lysis buffer (20 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH 7.4). Cells were lysed by 4 rounds of sonication, lasting 30 seconds each, with 30-second pauses in between, then centrifuged for 5 minutes at 10.000 g. The supernatant (comprising the cellular extract of E. coli BL21(DE3) and the induced pZA3 lCnLuc_his_tetR plasmid) was collected and used in the following tests. Each heat-inactivated recombinant K1F bacteriophage was then mixed with the cellular extract of E. coli BL21(DE3) harboring the induced pZA31CnLuc_his_tetR plasmid to produce a test sample. Figure 2 shows a significant increase in luminescence signal at 406 nm for each test sample comprising the recombinant K1F bacteriophages (compared to the empty LB medium), indicating the release of the fusion genes g6.7:NnLuc and gl4:NnLuc from each recombinant K1F bacteriophage head and the binding with CnLuc from the induced E. coli BL21(DE3).

[0131] The luminescence signals of similar test samples to those described in Figure 2 (i.e. test samples comprising KlFe6.7:NnLuc and KlFel4:NnLuc bacteriophages) were compared with a control (the cellular extract of E. Coli BL21(DE3) without pZA31CnLuc_his_tetR plasmid mixed with heat treated wild type (wt) K1F phage). Figure 3 shows that when mixed with the extract of BL21(DE3) + induced pZA31CnLuc_his_tetR, the heat-treated KlFe6.7:NnLuc and KlFel4:NnLuc recombinant bacteriophage samples produced luminescence signals that were significantly higher than the control. There is no increase in luminescence signal for test samples comprising the extract derived from E. Coli BL21(DE3) cells without pZA31CnLuc_his_tetR plasmid. Similarly, no increase in luminescence was seen if the BL21(DE3) + induced pZA31CnLuc_his_tetR extract was mixed with heat-inactivated K1F wt phage (i.e. intact KlFel4:NnLuc phage).

[0132] Test 1 concludes that the fusion proteins, g6.7:NnLuc and gl4:NnLuc, are successfully packaged into KlFe6.7:NnLuc or KlFel4:NnLuc recombinant bacteriophages, respectively, and these fusion proteins are capable of producing light when released from the bacteriophage capsid and bound to free CnLuc in the presence of

[0133] Nano-Gio® Luciferase Assay. Test 2 (In Vivo) - Endpoint luminescence measurements of bacteriophage-bacterial interactions

[0134] Test 2 demonstrates the use of the recombinant K1F bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc) for the detection of cognate bacteria expressing the CnLuc gene in a test sample (E. coli EV36 + pZA31CnLuc_his_tetR), according to embodiments of a diagnostic test of the present invention.

[0135] The recombinant K1F bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc) were each mixed with E. coli EV36 + pZA31CnLuc_his_tetR to form a test sample and the luminescence of the test sample measured after around 12 minutes. This method is in accordance with embodiments of the method of performing the diagnostic test of the invention.

[0136] Figure 4 shows that the luminescence signals were significantly higher for test samples comprising KlFe6.7:NnLuc and KlFel4:NnLuc when compared to the negative control (E. coli EV36 without pZA31CnLuc_his_tetR). The luminescence signal was indistinguishable from the negative control in the case of E. coli Nissle 1917 + pZA31CnLuc_his_tetR (i.e. a non-cognate host of K1F bacteriophages) when challenged with either recombinant K1F bacteriophage.

[0137] Test 2 shows the expected result, wherein the cognate host / target bacterium (i.e. E. coli EV36 covered by the KI capsule and carrying the induced pZA31CnLuc_his_tetR) binds to the recombinant bacteriophage (KlFe6.7:NnLuc or KlFel4:NnLuc) and allows the injection of the contents of the bacteriophage head into the host, leading to the binding of CnLuc with the injected NnLuc fusion protein. Thus, a luminesce signal is produced (when Nano-Gio® Luciferase Assay is added). Also as expected, the non-cognate host (i.e. E. coli Nissle 1917, covered by the K5 capsule and carrying the induced pZA31CnLuc_his_tetR) produced no luminescence signal when challenged with either recombinant K1F bacteriophage (KlFe6.7:NnLuc or KlFel4:NnLuc) as the binding with the bacteriophages is not possible, therefore disallowing the injection of the contents of the bacteriophage head.

[0138] Test 3 (In Vitro) - Kinetic luminescence measurements of bacteriophage-bacterial interactions

[0139] Test 3 demonstrates how quickly the recombinant K1F bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc) can detect the presence of its cognate host / target bacterium (E. coli EV36 covered by the KI capsule and carrying the induced pZA31CnLuc_his_tetR) (i.e. how quickly the embodiments of the diagnostic test of the invention can detect the presence or absence of the target bacterial strain).

[0140] E. coli EV36 harbouring pZA31CnLuc_his_tetR plasmid were mixed with each recombinant K1F bacteriophages (KlFe6.7:NnLuc or KlFel4:NnLuc) and the timer was started. To stop the reaction, Nano-Gio® Luciferase Assay was added to the test samples at regular time intervals (followed by 10 seconds of shaking) and the resulting luminescence of the test sample was measured. Similar test samples to those used in Test 2 were used.

[0141] Figure 5 shows that the luminescence signals of the test samples measured at about 7 minutes 50 seconds after the addition of either of the recombinant bacteriophages (KlFe6.7:NnLuc and KlFel4:NnLuc) were significantly higher for samples containing E. coli EV36 + pZA31CnLuc_his_tetR compared to the negative control (E. coli EV36 without pZA31CnLuc_his_tetR). The luminescence signals of the samples containing E. coli Nissle 1917 + pZA31CnLuc_his_tetR (i.e. the non-cognate host) were indistinguishable from the negative control. It is known that in the T7 bacteriophage family, the transcription / translation of late bacteriophage genes starts at 8 minutes or later, thus, the significantly increased luminescence signal measured at 7 minutes 50 seconds - 7 minutes 55 seconds marks the functionality of the NnLuc fusion proteins injected from the bacteriophage head. Figure 6 shows the effect KlFe6.7NnLuc on E. coli EV36 + induced pZA31CnLuc_his_tetR and E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR. A significant increase in luminescence was seen for the prior, compared to the latter, starting at 7 minutes 20 seconds.

[0142] Test 4 - Verifying the source of luminescence

[0143] Test 4 provides evidence that the luminescent signals detected in the luminescent tests of the examples are produced by the NnLuc fusion proteins injected / released from the bacteriophage head, as opposed to being transcription / translation products of the injected bacteriophage genome. Two experiments were conducted to verify this, the first repeated the kinetic measurements described above but included luminescence signal detection at an earlier time point (i.e. 5 minutes 45 seconds). The second experiment involved adding a transcription inhibitor (rifampicin) to the test samples to eliminate the possibility of any luminescence signals arising from NnLuc synthesised by the target cell.

[0144] In the first experiment, E. coli EV36 + induced pZA3 lCnLuc_his_tetR or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR were challenged with either KlFe6.7:NnLuc or KlFel4:NnLuc bacteriophages. After 5 minutes 45 seconds, Nano- Glo® buffer was added to lyse the cells. Then, after a 10-minute incubation period, the

[0145] Nano-Gio® substrate was added and the resulting luminescence signals of each test sample was recorded at 460 nm. Figure 7 shows that the luminescence signal detected upon challenge with either KlFe6.7:NnLuc (A) or KlFel4:NnLuc (B) bacteriophages was significantly higher when a cognate host (E. coli EV36) was present compared to when a non-cognate host (E. coli Nissle 1917) was present. As mentioned above, transcription / translation of late T7 bacteriophage genes (e.g. including g6.7 and gl4 orthologues) starts at 8 minutes or later (Molineux, I., 2006. The T7 group, p 277- 301., In Calendar, R., The bacteriophages. The Oxford University Press, Oxford, UK). Thus, this increased luminescence signal detected after 5 minutes 45 seconds provides further evidence for the functionality of the NnLuc fiision proteins injected / released from the bacteriophage head.

[0146] In the second experiment, E. coli EV36 + induced pZA3 lCnLuc_his_tetR or E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR were challenged with either KlFe6.7:NnLuc or KlFel4:NnLuc bacteriophages. After 4 minutes of incubation, 50 pg / mL of rifampicin was added to each test sample to stop transcription. Figure 8 shows that rifampicin had only a minor, if any, effect on the luminescence signal produced by E. coli EV36 + induced pZA31CnLuc_his_tetR detected at 6 minutes 30 seconds. Rifampicin caused a 26% decrease in luminescence signal for E. coli EV36 + induced pZA31CnLuc_his_tetR infected with KlFe6.7:NnLuc bacteriophage (as shown in Figure 8A), but there was no significant decrease in luminescence signal resulting from infection with KlFel4:NnLuc bacteriophage (as shown in Figure 8B). As shown in Figure 8, the luminescence signal plateaued for E. coli EV36 + induced pZA31CnLuc_his_tetR following addition of rifampicin, as opposed to the continuing increase in luminescence signal seen for E. coli EV36 + induced pZA3 !CnLuc_his_tetR that was not treated with rifampicin. Figure 8 also shows no significant luminescent signal for E. coli Nissle 1917 + induced pZA31CnLuc_his_tetR (non-cognate host), which indicates that the noncognate host does not allow the release of the fusion proteins from the bacteriophage head.

[0147] These experiments verify that the injection / release of a protein from the phage head into the target cell can be used to detect successful phage infection.

[0148] Diagnostic test strip

[0149] Figure 9 illustrates a further embodiment of the diagnostic test of the invention, wherein the diagnostic test comprises a test strip. This embodiment demonstrates how embodiments of the diagnostic test of present invention, such as those described in the example above may be translated for use in clinic, or for use at home.

[0150] In this embodiment, a bacteriophage is provided on a lateral flow test strip. Any reagents and substrates that are required to produce the signal are also provided on the test strip. When in use, the test sample is applied to the test strip and contacts the bacteriophage present on the test strip.

[0151] The test strip comprises at least one control signal “C” to indicate whether the test has been correctly performed, and a sample signal “S” which indicates the result of the test based on the test sample applied.

[0152] In use, the test strip will show a positive or negative result. A positive result will show if the bacteriophage contacts a test sample comprising its target bacterium and binds to its target bacterium. A negative result will show if the bacteriophage contacts a test sample that does not comprise its target bacterium and / or does not bind to its target bacterium. When the diagnostic test of Figure 9A is in use, a positive result results in appearance of a sample signal on the test strip, and a negative result results in the absence of a signal on the test strip. In the embodiment shown in Figure 9A, the sample signal comprises a plus (+) symbol. When the diagnostic test of Figure 9B is in use, a positive result results in the appearance of a first sample signal on the test strip, and a negative result results in the appearance of a second sample signal on the test strip. In the embodiment shown in Figure 9B, the first sample signal (i.e. positive result) comprises a plus (+) symbol and a colour, and the second sample signal (i.e. negative result) comprises a minus (-) symbol and a colour that is different to the first sample signal.

[0153] The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

[0154]

[0155] SEQ ID NO: 2 ( amino acid sequence of g!4 )

[0156] MCEPVSIGLGIMAVAGATMSASQQAKAEGAAIDAQNRQAQEMVKQMNYSDAN

[0157] LRMQERDLKEQQMAELTETTLNGIRNQGMVRAAVAESGLEGNSMDRIERQVEG

[0158] DTVKERAGITESYNRDYAAIFGNRIANIENTKSAIRGQGKIIKTSPLAHALNVANA

[0159] GMQGYAAGKSISGASSSGGSAPISAAKGTPTGHS

[0160] SEQ ID NO: 3 (fusion gene sequence, g6.7 :NnLuc)

[0161] ATGTGTTTCAGTCCGAAGATTAGCACTCCGAAGCCTTCGGTCCAAGCACCTG

[0162] AACCAGCACCTCTGAGTGAGGAAGTTGCGTCAGTTGACATCGGGGCTGAATC

[0163] GGATGTGGACACCAATGAGACCAAAGGTATCAAAGACCTTAAGGTCAAGAA

[0164] GGAGTCTGCACCTAAAGATAAATCGTCAGTTAGCCGCGCCATGCGAGCCTCT

[0165] GGCGTCAACATGGGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGTCTTCAC

[0166] ACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGAC

[0167] CAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTC

[0168] CGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATC

[0169] GACATCCATGTCATCATCCCGTATGAATAA SEQ ID NO: 4 (fusion gene sequence, g!4:NnLuc)

[0170] ATGTGCGAGCCAGTAAGTATCGGTCTAGGAATCATGGCTGTAGCAGGGGCCA

[0171] CTATGTCCGCATCTCAACAGGCCAAAGCTGAGGGTGCTGCTATCGACGCTCA

[0172] GAACCGACAGGCTCAGGAGATGGTTAAGCAGATGAATTACTCTGACGCCAAC

[0173] CTAAGGATGCAGGAGCGAGACCTTAAGGAACAGCAGATGGCTGAACTGACA

[0174] GAGACCACGTTAAACGGTATCCGCAATCAGGGCATGGTACGAGCTGCGGTAG

[0175] CTGAGTCCGGTCTGGAAGGAAACTCTATGGACAGGATTGAACGTCAGGTAGA

[0176] GGGAGATACAGTCAAGGAGAGAGCAGGGATTACCGAAAGTTACAACCGCGA

[0177] CTATGCGGCTATCTTTGGGAACCGTATCGCCAACATTGAGAACACCAAGTCT

[0178] GCTATCCGTGGTCAAGGTAAAATCATCAAGACTAGCCCACTGGCTCATGCAC

[0179] TTAATGTTGCTAACGCCGGTATGCAGGGATACGCTGCTGGTAAGTCAATCTCT

[0180] GGGGCATCAAGCTCTGGTGGTTCTGCACCGATTAGTGCTGCTAAAGGCACAC

[0181] CTACAGGTCATAGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGTCTTCACA

[0182] CTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACC

[0183] AAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCC

[0184] GTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCG

[0185] ACATCCATGTCATCATCCCGTATGAATAA

[0186] SEQ ID NO: 5 (pSBC3g6. 7:NnLuc)

[0187] ACTAGTAGCGGCCGCTGCAGCTCTACGCCGGACGCATCGTGGCCGGCATCAC

[0188] CGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGG

[0189] GAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTA

[0190] TGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGC ACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCT

[0191] TCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGC

[0192] CTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCC

[0193] GCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGC

[0194] GCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATC

[0195] GGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGT

[0196] CACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGC

[0197] ATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCT

[0198] GGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCC

[0199] GCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGC

[0200] TTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTG

[0201] ATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCAT

[0202] GGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGT

[0203] GCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAA

[0204] CGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTG

[0205] AATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCA

[0206] GCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCG

[0207] CATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTA

[0208] CTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTG

[0209] CTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGT

[0210] GTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCC

[0211] GGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGT

[0212] ATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCA

[0213] TCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCA TCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTAC

[0214] CCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAA

[0215] AAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTC

[0216] TGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAAT

[0217] CGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGT

[0218] GATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTT

[0219] GTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGG

[0220] GTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGG

[0221] AGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC

[0222] ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCAT

[0223] CAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC

[0224] TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA

[0225] ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC

[0226] CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC

[0227] CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG

[0228] ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT

[0229] CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG

[0230] GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG

[0231] GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC

[0232] GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA

[0233] CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT

[0234] GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA

[0235] GAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAA

[0236] AAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG

[0237] ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT

[0238] TAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT

[0239] AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG

[0240] TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT

[0241] ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC

[0242] GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACG

[0243] CTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG

[0244] CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG

[0245] CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT

[0246] GCCATTGCTGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT

[0247] CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC

[0248] AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG

[0249] CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC

[0250] ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATT

[0251] CTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGG

[0252] GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC

[0253] GTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG

[0254] ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG

[0255] CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAAT

[0256] AAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATT

[0257] GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT

[0258] TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

[0259] CTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCG TATCACGAGGCCCTTTCGTCTTCAAGAATTCGCGGCCGCTTCTAGAGGGTTTT

[0260] CAGCCCAGCGGAGTAAGCACTTTTAGCCAACCTAACGTCGCTACAGTAGCGG

[0261] CTGCACCTGAAGAGGAGACTCTAACTCCTCAACAGAAAGCTGCGCGTATTCG

[0262] TGCTGCGAACAGGGCCGATAAACTGGCTGAGTCCAACAACTGACTATCACTA

[0263] TAGGGATACCGTTCAGTTCGATTAACTGTAGGAGGGACTATGTGTTTCAGTCC

[0264] GAAGATTAGCACTCCGAAGCCTTCGGTCCAAGCACCTGAACCAGCACCTCTG

[0265] AGTGAGGAAGTTGCGTCAGTTGACATCGGGGCTGAATCGGATGTGGACACCA

[0266] ATGAGACCAAAGGTATCAAAGACCTTAAGGTCAAGAAGGAGTCTGCACCTA

[0267] AAGATAAATCGTCAGTTAGCCGCGCCATGCGAGCCTCTGGCGTCAACATGGG

[0268] GTCTGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTATGGTTT

[0269] TCACCCTGGAAGACTTCGTTGGTGACTGGCGTCAGACCGCTGGTTACAACCT

[0270] GGACCAGGTTCTGGAACAGGGTGGTGTTTCTTCTCTGTTCCAGAACCTGGGTG

[0271] TTTCTGTTACCCCGATCCAGCGTATCGTTCTGTCTGGTGAAAACGGTCTGAAA

[0272] ATCGACATCCACGTTATCATCCCGTACGAATAATTGAAACCCCTTGGGTGCCT

[0273] TCGGGTGCTTGAGGGGTTTTTGCTTAAAGTGAGAGGAGACTTATGGCTCAAT

[0274] ACATTCCACTGAATGCTAACGATGACTTAGATGCCATCAACGATATGTTAGCT

[0275] GCTATCGGTGAACCAGCAGTCCT

[0276] SEQ ID NO: 6 (pSBC3g!4:NnLuc)

[0277] ACTAGTAGCGGCCGCTGCAGCTCTACGCCGGACGCATCGTGGCCGGCATCAC

[0278] CGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGG

[0279] GAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTA

[0280] TGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGC ACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCT

[0281] TCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGC

[0282] CTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCC

[0283] GCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGC

[0284] GCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATC

[0285] GGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGT

[0286] CACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGC

[0287] ATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCT

[0288] GGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCC

[0289] GCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGC

[0290] TTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTG

[0291] ATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCAT

[0292] GGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGT

[0293] GCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAA

[0294] CGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTG

[0295] AATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCA

[0296] GCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCG

[0297] CATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTA

[0298] CTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTG

[0299] CTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGT

[0300] GTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCC

[0301] GGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGT

[0302] ATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCA

[0303] TCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCA TCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTAC

[0304] CCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAA

[0305] AAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTC

[0306] TGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAAT

[0307] CGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGT

[0308] GATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTT

[0309] GTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGG

[0310] GTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGG

[0311] AGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC

[0312] ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCAT

[0313] CAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC

[0314] TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA

[0315] ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC

[0316] CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC

[0317] CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG

[0318] ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT

[0319] CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG

[0320] GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG

[0321] GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC

[0322] GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA

[0323] CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT

[0324] GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA

[0325] GAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAA

[0326] AAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG

[0327] ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT

[0328] TAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT

[0329] AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG

[0330] TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT

[0331] ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC

[0332] GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACG

[0333] CTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG

[0334] CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG

[0335] CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT

[0336] GCCATTGCTGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT

[0337] CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC

[0338] AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG

[0339] CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC

[0340] ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATT

[0341] CTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGG

[0342] GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC

[0343] GTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG

[0344] ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG

[0345] CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAAT

[0346] AAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATT

[0347] GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT

[0348] TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

[0349] CTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCG TATCACGAGGCCCTTTCGTCTTCAAGAATTCGCGGCCGCTTCTAGAGGGTTTT

[0350] CAGCCCAGCGGAGTAAGCACTTTTAGCCAACCTAACGTCGCTACAGTAGCGG

[0351] CTGCACCTGAAGAGGAGACTCTAACTCCTCAACAGAAAGCTGCGCGTATTCG

[0352] TGCTGCGAACAGGGCCGATAAACTGGCTGAGTCCAACAACTGACTATCACTA

[0353] TAGGGATACCGTTCAGTTCGATTAACTGTAGGAGGGACTATGTGTTTCAGTCC

[0354] GAAGATTAGCACTCCGAAGCCTTCGGTCCAAGCACCTGAACCAGCACCTCTG

[0355] AGTGAGGAAGTTGCGTCAGTTGACATCGGGGCTGAATCGGATGTGGACACCA

[0356] ATGAGACCAAAGGTATCAAAGACCTTAAGGTCAAGAAGGAGTCTGCACCTA

[0357] AAGATAAATCGTCAGTTAGCCGCGCCATGCGAGCCTCTGGCGTCAACATGGG

[0358] GTCTGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTATGGTTT

[0359] TCACCCTGGAAGACTTCGTTGGTGACTGGCGTCAGACCGCTGGTTACAACCT

[0360] GGACCAGGTTCTGGAACAGGGTGGTGTTTCTTCTCTGTTCCAGAACCTGGGTG

[0361] TTTCTGTTACCCCGATCCAGCGTATCGTTCTGTCTGGTGAAAACGGTCTGAAA

[0362] ATCGACATCCACGTTATCATCCCGTACGAATAATTGAAACCCCTTGGGTGCCT

[0363] TCGGGTGCTTGAGGGGTTTTTGCTTAAAGTGAGAGGAGACTTATGGCTCAAT

[0364] ACATTCCACTGAATGCTAACGATGACTTAGATGCCATCAACGATATGTTAGCT

[0365] GCTATCGGTGAACCAGCAGTCCT

[0366] SEQ IN NO: 7 (DNA sequence of flexible linker:NnLuc)

[0367] TCTGGTGGCGGTTCTGGTGGCGGTGGCTCTGGCGGTGGCGGTTCTATGGTT

[0368] TTCACCCTGGAAGACTTCGTTGGTGACTGGCGTCAGACCGCTGGTTACAAC

[0369] CTGGACCAGGTTCTGGAACAGGGTGGTGTTTCTTCTCTGTTCCAGAACCTG

[0370] GGTGTTTCTGTTACCCCGATCCAGCGTATCGTTCTGTCTGGTGAAAACGGT

[0371] CTGAAAATCGACATCCACGTTATCATCCCGTACGAA SEQ ID NO: 8 (DNA sequence of CnLuc)

[0372] ATGGGTGGTCTGTCTGGTGACCAGATGGGTCAGATCGAAAAAATCTTCAAAG

[0373] TTGTTTACCCGGTTGACGACCACCACTTCAAAGTTATCCTGCACTACGGTACT CTGGTTATCGACGGTGTTACCCCGAACATGATCGACTACTTCGGTCGTCCGTA

[0374] CGAAGGTATCGCTGTTTTCGACGGTAAAAAAATCACCGTTACCGGTACTCTGT

[0375] GGAACGGTAACAAAATCATCGACGAACGTCTGATCAACCCGGACGGTTCTCT

[0376] GCTGTTCCGTGTTACCATCAACGGTGTTACCGGTTGGCGTCTGTGCGAACGTA

[0377] TCCTGGCT

Claims

CLAIMS1. A diagnostic test for detecting the presence or the absence of at least one target bacterium in a test sample, the diagnostic test comprising at least one bacteriophage specific to the or each target bacterium, wherein the or each bacteriophage comprises at least one signal molecule within or encapsulated in the head of the or each bacteriophage, and wherein the or each signal molecule is configured to be released from the head of the bacteriophage and produce a signal that can be detected upon the infection of the or each target bacterium with the or each bacteriophage, when the or each target bacterium is present in a test sample, in use.

2. A diagnostic test as claimed in claim 1 , wherein the or each bacteriophage is a replication defective bacteriophage.

3. A diagnostic test as claimed in claim 1 or 2, wherein at least one bacteriophage is from the T7 bacteriophage family and the target bacterium is E.Coli.

4. A diagnostic test as claimed in any preceding claim, wherein the or each signal molecule is independently selected from the group consisting of: a colourchanging enzyme, a light-emitting enzyme, a part or parts of a light-emitting split-enzyme, a part or parts of a colour-changing split-enzyme, a small molecule dye, a nucleic acid intercalating dye, and any combination thereof.

5. A diagnostic test as claimed in any preceding claim, wherein the or each signal molecule has a molecular weight of between 0.1 kDa and 10 kDa, or more preferably between 0.1 kDa and 5 kDa.

6. A diagnostic test as claimed in any preceding claim, wherein at least one signal molecule is fused to at least one internal capsid protein of the or each bacteriophage.

7. A diagnostic test as claimed in any preceding claim, wherein at least one signal molecule is fused to at least one leader peptide of the or each bacteriophage.

8. A diagnostic test as claimed in any preceding claim, wherein at least one signal molecule is one or more part of a split-enzyme, and wherein said one or more part of the split-enzyme is within or encapsulated in the head of at least one bacteriophage, and wherein the diagnostic test further comprises the complementary part or parts of said split-enzyme provided outside of the or each bacteriophage, and wherein the split-enzyme is capable of producing a signal upon binding of the parts of the split-enzyme following the release of the one or more part of the split-enzyme from the head of the or each bacteriophage.

9. A diagnostic test as claimed in claim 8, wherein the split-enzyme comprises a smaller part and a larger counterpart, and wherein the smaller part of the splitenzyme is within or encapsulated in the head of the or each bacteriophage and the larger counterpart of the split-enzyme is provided outside of the or each bacteriophage.

10. A diagnostic test as claimed in claim 8 or 9, wherein the or each bacteriophage comprises a part of a split-nanoluciferase that is within or encapsulated in the head of the or each bacteriophage, and wherein the diagnostic test further comprises the complementary part of said split-nanoluciferase provided outside of the or each bacteriophage.

11. A diagnostic test as claimed in any preceding claim, wherein the diagnostic test further comprises at least one reagent and / or substrate required by the or each signal molecule to generate the signal, and wherein the or each reagent and / or substrate is provided outside the or each bacteriophage.

12. A diagnostic test as claimed in claim 10, wherein the diagnostic test further comprises the substrate furimazine or equivalent photoluminescent reagent.

13. A diagnostic test as claimed in any preceding claim, the diagnostic test further comprises a test strip and wherein the test trip comprises the or each bacteriophage.

14. A diagnostic test as claimed in any preceding claim, wherein the diagnostic test detects the presence or the absence of the or each target bacterium in a test sample in less than 15 minutes, 10 minutes, 7 minutes, 5 minutes, or less than 2 minutes.

15. A diagnostic test as claimed in any preceding claim for use in medical testing, for the detection of the presence or absence of at least one bacterial infection in a test sample.

16. The use of a diagnostic test as claimed in any one of claims 1-14 for the detection of the presence or absence of bacterial contamination in a food or beverage product.

17. The use of a diagnostic test as claimed in any one of claims 1-14 for the detection of the presence or absence of bacterial contamination in a water sample.

18. A method of detecting at least one bacterium, the method comprising:(i) providing a diagnostic test comprising at least one bacteriophage specific to at least one bacterium, and wherein the or each bacteriophage comprises at least one signal molecule within or encapsulated in the head of the or each bacteriophage, and wherein the or each signal molecule is configured to be released from the head of the bacteriophage and thereby produce a signal that can be detected upon the infection of at least one target bacterium with the or each bacteriophage, when the or each target bacterium is present in a test sample, in use;(ii) contacting the or each bacteriophage with a test sample;(iii) incubating the at least one bacteriophage with the test sample; and(iv) detecting the presence or absence of a signal from the at least one signal molecule, wherein the presence of a signal indicates the presence of at least one bacterium specific to the or each bacteriophage in the test sample.

19. A method as claimed in claim 18, wherein step (i) further comprises providing at least one reagent and / or substrate required by the or each signal molecule to produce a signal in the presence of the or each bacterium.

20. A method as claimed in claim 18 or 19, wherein the or each signal molecule produces a light and / or colour signal and step (iv) comprises detecting said light and / or colour signal.

21. A method of encapsulating a signal molecule inside the head of a bacteriophage, the method comprising engineering the bacteriophage with at least one fusion gene, wherein the or each fusion gene comprises a gene encoding an internal capsid protein of the bacteriophage genome and a gene encoding a signal molecule.

22. A method as claimed in claim 21, wherein the method comprises directly engineering the bacteriophage genome to incorporate the or each fusion gene, wherein the direct engineering of the bacteriophage comprises selecting a gene encoding an internal capsid protein from the bacteriophage genome and fusing to it a gene encoding a signal molecule.

23. A method as claimed in claim 21, wherein the method comprises removing from the bacteriophage genome, or supressing, the gene encoding the internal capsid protein and supplying said gene to the bacteriophage as part of the or each fusion gene via at least one vector.

24. A method of encapsulating a signal molecule inside the head of a bacteriophage via a cell-free expression system, the method comprising contacting a cell-free preparation with the genome of a bacteriophage and atleast one fusion gene in a cell-free expression reaction, wherein the or each fusion gene comprises a gene encoding an internal capsid protein and a gene encoding a signal molecule.

25. A method of encapsulating a small molecule inside the head of a bacteriophage via a cell-free expression system, the method comprising contacting a cell-free preparation with the genome of a bacteriophage in a cell- free expression reaction and providing a small molecule to the cell-free expression reaction, and wherein the small molecule is a signal molecule or a substrate molecule for a signal molecule.

26. A method as claimed in claim 25, wherein the small molecule has a molecular weight of between 100 g mol1- 1400 g mol1, or more preferably, 100 g mol1- 1000 g mol1.

27. A bacteriophage formed by the method of any one of claims 21-26 for use in the diagnostic test of any one of claims 1-14.

Images