WO2006105504A1

WO2006105504A1 - Apparatus and method for detecting microorganisms using flagged bacteriophage

Info

Publication number: WO2006105504A1
Application number: PCT/US2006/012371
Authority: WO
Inventors: Jon Carlton Rees
Original assignee: Microphage Incorporated
Priority date: 2005-03-31
Filing date: 2006-03-31
Publication date: 2006-10-05
Also published as: WO2006105414A3; US20070059725A1; WO2006105414A2; US8092990B2

Abstract

AA method of determining the presence or absence of a target microorganism in a test sample, the method comprising: flagging a bacteriophage (70) with a detectable flag; combining flagged bacteriophage with the sample and providing conditions sufficient to allow flagged bacteriophage to attach to the microorganism creating a bacteriophage exposed sample; performing a target separation process, the process capable of separating the bacteriophage exposed sample into a target microorganism portion containing target microorganisms present in the sample and a portion containing flagged bacteriophage not bound to the target bacteria; and assaying at least one of the portions to detect the presence or absence of the flag to determine the presence or absence of target microorganisms in the sample. The flag (90) can be colloidal gold particles, lectins, aptomers, immunoadhesion molecules, biotin molecules, latex beads, fluorophores or other color-based molecule, and the like. The invention also provides a lateral flow strip (140) having a conjugate pad (143) containing flagged bacteriophage (142) and an immobilization zone (146) containing capture elements (170) specific to the target bacteria.

Description

APPARATUS AND METHOD FOR DETECTING MICROORGANISMS USING FLAGGED BACTERIOPHAGE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of detection of microscopic living organisms, and more particularly to the detection of bacteria utilizing bacteriophage.

2. Statement of the Problem Bacteriophage are viruses that by definition specifically infect bacteria, and in the case of lytic bacteriophage, rapidly kill the host bacterium while producing progeny phage particles. Bacteriophage have been observed to often be specific for a species or strain of bacteria, that is, a bacteriophage particle may only infect a group of bacteria that are phylogenetically related. Because of this specificity for strains of bacteria, attempts have been made to exploit bacteriophage for diagnostic purposes, such as the identification of bacteria in test samples. Further, since bacteriophage multiply within minutes or hours to provide thousands of additional phage that can be detected, it has been observed that the multiplied bacteriophage may be much easier to detect than the bacteria themselves. This is referred to bacteriophage amplification. Thus, bacteria detection methods using bacteriophage have shown promise to be faster and more sensitive than conventional bacteria identification methods. See, for example, United States Patent Publication No. US-2005-0003346- A1 , which is hereby incorporated by reference to the same extent as though fully disclosed herein. Bacteriophage infection processes have been well elucidated, typically comprising of a binding event in which the phage attaches itself to a target bacterium, injection of the nucleic acid into the host bacterium, generation of phage proteins and nucleic acid molecules, assembly of the phage particles, and lysis of the bacterial host thereby releasing progeny bacteriophage into the surrounding medium. Thus, phage- based diagnostics described throughout the literature rely upon the detection of phage-linked metabolic activity to imply the presence of the targeted bacteria. A comprehensive review, Mandeville, R.; Griffiths, M.; Goodridge, L.; Mclntyre, L.; Ilenchuk, T. T.; Diagnostic and Therapeutic Applications of Lytic Phages, 2003 Anal. Lett., 36, 15, 3241-3259, has organized phage-based diagnostic tests into the following categories:

• Detection of Intracellular Components Released During Phage Lysis

• Detection Based on Inhibition of Metabolism and Growth

• Detection of (amplified) Phage Particles

• Phage as Staining Agents

• Phage as Transducing Agents: The Reporter Phage Approach • Luciferase Reporter Systems: Prokaryotic Luciferase

• Luciferase Reporter Systems: Eukaryotic Luciferase

• Luciferase Applications

• Indirect Detection Applications

United States Patent Publications No. 2002/0127547 and No. 2004/0121403 in the name of Stefan Miller describing a diagnostic assay in which whole phage or phage proteins are bound to a support, the support is immersed in a sample to be tested, the phage are allowed to capture target bacteria in the sample, the support is removed from the sample and unbound materials are washed off, and a diagnostic assay is performed to detect either the bacteria or the phage proteins.

All of the above methods require time and conditions for bacteriophage metabolic activity, such as reproduction and bacteria lysis, to proceed and/or other complex and time consuming diagnostic processes. Thus, bacteriophage-based methods of detecting microorganisms have yet to demonstrate the promise they hold. What is needed is a bacteriophage detection method combining the specificity to particular microorganisms that bacteriophage provide with simpler and faster diagnostic processes.

SUMMARY OF THE INVENTION The invention solves the above problems, as well as other problems of the prior art, by providing a faster method of microbial detection using bacteriophage. The invention recognizes that phage specificity is due in specific part to the phage binding event that initiates phage infection. As known in the art, this binding event takes place at specific receptor sites on the surface of the microorganism to which phage specific proteins bind, e.g., lipopolysaccharides, cell wall proteins, teichoic acid, flagellar proteins, pilus proteins, etc. Once the end plate of the phage settles onto the surface of the microorganism, a conformational change takes place in the phage that forces the tail fibers or core through the bacterial cell wall, thereby attaching the phage to the surface of the microorganism and is commonly recognized as an irreversible event. This typically occurs within one to two minutes of phage-microorganism interaction. The invention harnesses this bacteriophage property to provide microbial diagnostic apparatus and processes that incorporate only the phage binding event of the infection cycle, which can take just a few minutes. The invention preferably provides detection methods and apparatus that directly utilize the phage binding event, and do not require waiting for the phage amplification process or other phage metabolic processes to complete their cycles. The invention provides a method of determining the presence or absence of a target microorganism in a sample to be tested, the method comprising: (a) flagging a bacteriophage with a detectable flag; (b) combining the flagged bacteriophage with the sample and providing conditions sufficient to allow the flagged bacteriophage to bind to the microorganism to create a bacteriophage exposed sample; (c) performing a target separation process, the separation process capable of separating the bacteriophage exposed sample into a target microorganism portion containing target microorganisms present in the sample and an unbound flagged bacteriophage portion containing flagged bacteriophage that are not bound to the target microorganism; and (d) assaying at least one of the portions to detect the presence or absence of the flag to determine the presence or absence of the target microorganism in the sample. Preferably, the microorganism is a bacterium and the assaying comprises detecting the flag as an indication of the presence of the target bacterium in the sample. Preferably, the combining comprises applying the sample to a lateral flow strip. Preferably, the performing a target separation process comprises capturing the flagged and bound phage with a capture element specific to the target microorganism. Preferably, the flagging comprising attaching to the bacteriophage a detectable element selected from the group consisting of: gold particles, lectins, aptomers, immunoadhesion molecules, biotin molecules, polystyrene beads, latex beads, fluorophores and other color-based molecules. Preferably, the assaying comprises a method selected from the group consisting of: detecting color, detecting a shade, detecting fluorescence, detecting luminescence, immuno-detection, electrical detection and magnetic detection. Alternatively, the assaying comprises detection with an electrical, magnetic or optical instrument. Preferably, the assaying comprises determining the concentration of the target microorganism in the sample. Preferably, the performing a target microorganism separation process comprises washing the exposed sample. Preferably, the washing comprises centrifugation and decanting the supernatant. Preferably, the washing comprises filtering the bacteriophage exposed sample through a filter that allows unbound phage to pass through while retaining the bound phage. Preferably, the washing comprises rinsing with a wash solution. Preferably, the rinsing comprises rinsing with a low salt, neutral pH, or tris-based buffer. Preferably, the sample is a fluid and the combining comprises pouring the sample into a container containing the flagged bacteriophage. Preferably, the performing a target separation process comprises diffusion of the bacteriophage. Preferably, the combining comprises adding the sample to one of a plurality of chambers in a vial. The invention also provides apparatus for detecting a target microorganism, the apparatus comprising: a substrate; a conjugate pad on or in the substrate, the conjugate pad containing bacteriophage conjugated with a detectable flag, the bacteriophage specific to the target microorganism; and an immobilization zone on or in the substrate, the immobilization zone including an immobilization element designed to immobilize the target microorganism. Preferably, the immobilization zone comprises antibodies, aptamers, or bacteriophage. Preferably, the flag comprises a colored element. Preferably, the flag comprises a conductive element. Preferably, the flag comprises gold. Preferably, the flag comprises a magnetic element. Preferably, substrate comprises a lateral flow strip. Preferably, the microorganism is a bacterium. Preferably, the apparatus further comprises an internal control zone. In another aspect, the invention provides apparatus for detecting a target microorganism, the apparatus comprising a flagged bacteriophage and a filter.

In a further aspect, the invention provides apparatus for detecting a target microorganism, the apparatus comprising a flagged bacteriophage and a separation element that permits the flagged bacteriophage to pass but does not allow the target microorganism to pass. Preferably, separation element is selected from the group consisting of: a filter and a membrane. Preferably, the apparatus further comprises a vial having two chambers, with the separation element separating the two chambers.

In yet another aspect, the invention provides a method of manufacturing a microbial test apparatus, the method comprising: providing a substrate and a biological material capable of attaching to a target microorganism; forming a line of the biological material on the substrate; and cutting the substrate in a direction essentially perpendicular to the line to form the test substrate. Preferably, the method further comprises forming a line of bacteriophage conjugated with a detectable flag, the line of bacteriophage being parallel to the line of biological material. Preferably, the substrate is a porous membrane. Preferably, the biological material is an antibody. Preferably, the providing comprises providing a first biological material and a second biological material, and the forming comprises forming a first line with the first biological material and a second line with the second biological material, with the first line and the second line being substantially parallel.

The invention provides bacteriophage-based microbial detection methods that provide a direct indication of the presence of a microbe. Typically, the detection methods according to the invention require less than twenty minutes, preferably less than fifteen minutes and most preferably, less than ten minutes. Numerous other features, objects, and advantages of the invention will become apparent from the following description when read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an illustration of a flagged bacteriophage;

FIG. 2 is a side cross-sectional view of a lateral flow device according to the invention;

FIGS. 3A, 3B, 3C and 3D illustrate the operation of the lateral flow device of FIG. 2;

FIGS. 4A, 4B and 4C illustrate an alternative method of microbe detection using flagged bacteriophage;

FIG. 5 illustrates a modification of the method of FIGS. 4A-4C in which the bacteriophage are not flagged until after they have infected the bacteria; and

FIGS. 6A and 6B illustrate another alternative preferred embodiment of the method according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The method of the invention relies on the usage of bacteriophage, or simply phage, to detect the presence of target microscopic living organism (microorganism), such as a bacterium, in a sample. In this disclosure, the terms "bacteriophage" and

"phage" include bacteriophage, phage, mycobacteriophage (such as for TB and paraTB), mycophage (such as for fungi), mycoplasma phage or mycoplasmal phage, and any other term that refers to a virus that can invade living bacteria, fungi, mycoplasmas, protozoa, and other microscopic living organisms and uses them to replicate itself. Here, "microscopic" means that the largest dimension is one millimeter or less. Bacteriophage are viruses that have evolved in nature to use bacteria as a means of replicating themselves. A phage does this by attaching itself to a bacterium and injecting its NUCLEIC ACID into that bacterium, inducing it to replicate the phage hundreds or even thousands of times. Some bacteriophage, called lytic bacteriophage, rupture the host bacterium, releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for phage infection of a bacterium, phage multiplication or amplification in the bacterium, to lysing of the bacterium takes anywhere from thirty minutes to hours, depending on the phage and bacterium in question and the environmental conditions. The prior art methods of bacteriophage-based microbial detection generally depend on this bacterial lysing process. The methods and apparatus of the invention do not require the lysing, so can be applied to more varieties of bacteriophage. Since they do not require the incubation time, the methods and apparatus of the invention are also faster than prior art methods.

The disclosed detection method offers a combination of specificity, sensitivity, simplicity, speed, and/or cost which is superior to any currently known microscopic organism detection method. The method taught herein relies on the usage of bacteriophage to directly detect the presence of one or more target bacterium in a sample. A typical bacteriophage 70, in this case T4, is shown in FIG. 1. Structurally, a bacteriophage 70 comprises a protein shell or capsid 72, sometimes referred to as a head, that encapsulates the viral nucleic acids 74, i.e., the nucleic acid and/or RNA. A bacteriophage may also include internal proteins 75, a neck 76, a tail sheath 77, tail fibers 78, an end plate 79, and pins 80. The capsid 72 is constructed from repeating copies of one or more proteins. In the methods and apparti of the invention, the bacteriophage of interest are flagged or labeled with some type of flagging device 90 that can easily be detected by the human eye or standard instrumentation. For example, many studies show the feasibility of attaching colloidal gold, fluorphores, small molecules, and even larger biopolymers to proteinaceous moieties. Examples of flagging agents are colloidal gold particles, lectins, aptomers, immunoadhesion molecules, biotin molecules, particularly those using the succinimide esters of biotin, polystyrene beads, latex beads, fluorophores or other color-based molecule, and the like. Flagging agents can also be magnetic particles. When the flagged bacteriophage attach themselves to a target microbe, such as a target bacteria, the target microbe is in turn flagged for immediate and direct detection. If a target bacteria separation process capable of separating the bacteriophage exposed sample into a target bacteria portion containing target bacteria present in the sample and an unbound flagged bacteriophage portion containing flagged bacteriophage that are not bound to the target bacteria, then at least one of the separated portions can be assayed to detect the presence or absence of the flag to determine the presence or absence of the target microorganism in the sample.

Thus, the detection method according to the invention can take place in the time it takes for the bacteriophage to attach to the microbes, which is typically one to ten minutes. Once a flagged phage is bound to a target microorganism, the flag is exposed on the exterior of the microorganism. Typically several hundred to several thousand phage will interact and remain bound to the exterior of each microorganism thereby providing a large number of flag signals per microorganism. This large number of flags per microorganism enhances the detection process.

Microbiologists have isolated and characterized many thousands of phage species, including specific phages for most human bacterial pathogens. Individual bacteriophage species exist that infect bacterial families, individual species, or even specific strains. Table 1 lists some such phages and the bacterium they infect.

Table 1

This invention takes advantage of the existing characteristics of bacteriophage, such as highly specific phage-bacterial infection, resulting in a bacterial detection method which is highly specific to target bacteria, very sensitive, fast, simple to perform, and/or can be quite economical. Moreover, unlike other phage-based bacterial detection methods, the preferred method described does not depend on bacterial lysing and does not require the complex diagnostic techniques usually associated with this lysing. This dramatically reduces the time and costs associated with developing specific bacterial tests utilizing this method. 2. Detailed Description

It is straightforward to label, i.e., flag, a bacteriophage with some type of molecule that can easily be detected by the human eye or standard instrumentation if the molecules can be concentrated at a particular location. For example, many studies show the feasibility of attaching colloidal gold, fluorphores, small molecules, and even larger biopolymers to proteinaceous moieties. As the protective capsid of bacteriophage is comprised almost solely of protein, these proven chemistry techniques can be employed to attach various molecular labels to bacteriophage. For example, bacteriophage can be labeled with the small molecule biotin using the succinimide esters of biotin. Once a phage has been flagged, fairly uncomplicated procedures may be used to detect the binding of the phage to a bacterial substrate. In general, the steps of most procedures would be adding labeled phage to a bacterial sample, allowing the phage to bind to the bacteria, separation of bound phage from unbound phage, for example, by washing, and detection of the bound phage particles. In the preferred embodiment, phages, which are preferably specific to a target microorganism, are flagged with a detectable element 90, preferably, colloidal gold particles, polystyrene beads, or latex beads, and the labeled or flagged phage are incorporated into a lateral flow device 140. A cross-sectional view of the lateral flow strip 140 is shown in FIG. 2. The lateral flow strip 140 preferably includes a sample application pad 141 , a conjugate pad 143 which contains the labeled phage, a substrate 164 in which a detection line 146 and an internal control line 148 are formed, and an absorbent pad 152, all mounted on a backing 162, which preferably is plastic. The substrate 164 is preferably a porous mesh or membrane. It is made by forming lines 143, 146, and optionally line 148, on a long sheet of said substrate, then cutting the substrate in a direction perpendicular to the lines to form a plurality of substrates 164.

The conjugate pad 143 contains phage 70 each of which has been conjugated to a colloidal gold particle 90, forming phage-particle conjugates 142. Detection line 146 and control line 148 are both capture lines and each form an immobilization zone; that is, they contain a material that interacts in an appropriate way with the target bacteria to be detected. In the preferred embodiment, the interaction is one that immobilizes the bacteria and also the bacteriophage-particle conjugate. The capture molecules may be antibodies raised against bacteria, aptamers, or even phage particles themselves. Thus, detection line 146 comprises immobilized capture elements, with capture line 146 perpendicular to the direction of flow along the strip, and being dense enough to capture a significant portion of the bacteria in the flow. The capture elements are preferably specific to the target microorganism; that is, the capture elements essentially capture only a specific target bacteria. Optionally, strip 140 may include a second capture line 148 (FIG. 2) including a second capture element The second capture element may or may not be identical to the first capture element 170. Second capture line 148 may serve as an internal control zone to test if the assay functioned properly.

One or more drops of a test sample containing bacteria 310 are added to the sample pad 141 as shown in FIG 3A. The test sample flows along the lateral flow strip 140 toward the absorbent pad 152 at the opposite end of the strip. As the bacteria flow through the conjugate pad 143 toward the membrane, they pick up one or more of the phage-particle conjugates 142 forming phage-particle-bacteria complexes 154 as shown in FIG. 3B. In FIG. 3C, the phage-particle-bacteria complexes 154 flow toward capture line 146. As the phage-bead complexes move over capture line 146, they form an immobilized and concentrated phage-particle- bacteria-capture element complex 58 as shown in FIG. 3D. If enough phage-particle- bacteria complexes bind to the row 146 of immobilized capture elements, a gold- colored line 159 becomes visible to the naked eye. A visible line 159 indicates that the target bacteria were present in the raw sample. If the particle-labeled phage do not bind to any target bacteria, then no such line ever forms. If no line is formed, then target bacteria were not present in the raw sample or were present in concentrations too low to be detected with the lateral flow strip 140.

FIGS. 4A-4C illustrate a generalize procedure for directly identifying a target bacteria 410 using phage-detectable element complexes 142. A simple incarnation of the assay involves labeling bacteriophage 70 with a flag 90 such as a fluorophore or some color based molecule. The bacteriophage-detectable element complexes 142 are then be added to a sample containing a targeted bacterium 410 as shown in FIG. 4A. After the phages have been allowed enough time to bind to any targeted bacterium, as shown in FIG. 4B, a preferential washing process 460 can then be employed that leaves the phage-bacterium complex in a state that can be easily assayed, while washing away exogenous phage. Examples of such a washing could be centrifugation and decanting the supernatant, and filtering the sample through a filter 420 that allows unbound phage to pass through while retaining the phage- bacteria complex. Washing can also involve a thorough rinsing with a wash solution, such as a low salt, neutral pH, tris-based buffer. The phage-bacteria complex can then be subjected to a detection strategy as indicted in FIG. 4C, such as fluorescent excitation, visible color detection, or whatever strategy allows for the label to be detected.

A modification of the process of FIGS.4A-4C that may not require a pre-labeled bacteriophage is shown in FIG. 5. For example, if an immuno-detection platform is available that can detect the presence of phage, it may be employed to detect a bacteriophage bound to a bacterium as opposed to detection of a label attached to a bound bacteriophage. A phage-based bacterial detection scheme as described in the previous paragraph may be used. Phage and bacteria can be admixed, and time allowed for the phage to bind to a targeted bacterium. After centrifugation or filtration and washing, the bacteria-phage complex can be subjected to detection by an ascribed immuno-detection platform 540. If the immuno-detection strategy 540 returns a positive result for the presence of phage, then one can be assured that it could only be there because the phage has been bound to the bacteria and retained in the phage-bacteria complex. Thorough washing greatly mitigates the potential of false positives. Another embodiment of the invention is illustrated in FIGS. 6A and 6B. This embodiment is driven by a diffusion gradient caused by binding of phage to a target bacterium. A vial is 650 constructed that has two chambers 652 and 654 separated by a filter, 620, which is preferably a 0.2 micron filter 620 but also can be a membrane or other suitable separation element that allows bacteriophage to pass but does not allow bacteria to pass. In particular, a 0.2 micron filter does not allow bacteria to pass through its pores, but phage can freely pass through the pores driven by diffusive processes. The medium within the vial has labeled phages, for example bacteriophage 70 labeled with gold particles 90 forming bacteriophage-detectable element complexes 142, that are allowed to freely diffuse through the 0.2 micron filter. As bacteria 610 are added to one of the chambers of the vial, the phages begin the process of binding to the bacteria. As the phage in the bacteria side 654 of the vial bind to the targeted bacterium, the effective concentration of freely diffusive phage particles is decreased, and phage from the portion 652 of the vial where bacteria are not present diffuse across the membrane 620 where they also have the potential to bind to the bacteria 610, further increasing the numbers of phage that are able to diffuse to the bacteria side of the vial. A concentration of gold-labeled phage on the bacteria side 654 of the vial develops, deepening the gold color present in this side of the vial, while lightening the color in the bacteria-free side 652 of the vial. This color change may be visible to the eye, or may be detected by instrumentation. It is important to realize that if non-target bacteria are present in the bacterial side of the vial, then phage do not bind to the bacteria. Thus, no concentration gradient is created, and no apparent color change would be visible.

Any of the above processes according to the invention can be implemented using known standard cultures of bacteria. That is, a culture with a specific concentration when exposed to a specific phage-detection element conjugate result in a specific shade or color. Standard coloration or shade charts can be constructed which can be compared with the result of a test to determine the concentration of bacteria. Similarly, concentration can be determined by comparing the level of any other detectable parameter, such as an electrical parameter, a magnetic parameter or a level of fluorescence. It is also evident that different phage may be flagged with different detectable elements permitting tests for a plurality of different bacteria to be run simultaneously on a given sample.

In any of the above examples, the detection can be performed by an instrument. Scanning electron microscopes (SEM) can be used to identify thousands of flagged phage on a single bacterium, thus a SEM can be used in any of the embodiments above. As another example, an electrical instrument can be used to detect gold particles using electrical resistance or any other electrical parameter. If the flag is a magnetic particle, magnetic detection instruments may be used. An optical instrument may be used to detect color, shade, luminescence and fluorescence. In any of the above examples, after the target microorganism portion and the unbound flagged microorganism portion have been separated, either of the separated portions can be assayed to determine the presence of absence of the microorganism in the sample. Usually this will be by looking for the flag in the target microorganism portion, but in some cases the unbound flagged bacteriophage portion may be assayed. The absence of any unbound flagged bacteriophage in this portion can be indicative of the presence of a large number of the target microorganism, or a very high density of unbound flagged bacteriophage in this portion can be indicative of the absence of the target microorganism in the sample, or that the concentration of target microorganism is at a low level.

There has been described a microorganism detection method which is specific to a selected organism, sensitive, simple, fast, and/or economical, and having numerous novel features. This specificity is primarily due to the specificity of the phage binding to the target microorganism. The invention can be used in a wide variety of applications including human clinical diagnostics, veterinary diagnostics, food pathogen detection, environmental testing, and biowarfare detection. It should be understood that the particular embodiments shown in the drawings and described within this specification are for purposes of example and should not be construed to limit the invention, which will be described in the claims below. Further, it is evident that those skilled in the art may now make numerous uses and modifications of the specific embodiment described, without departing from the inventive concepts. Equivalent structures and processes may be substituted for the various structures and processes described; the subprocesses of the inventive method may, in some instances, be performed in a different order; or a variety of different materials and elements may be used. Consequently, the invention is to be construed as embracing each and every novel feature and novel combination of features present in and/or possessed by microorganism detection apparatus and methods described.

Claims

CLAIMS We claim:

1. A method of determining the presence or absence of a target microorganism in a sample to be tested, said method comprising: (a) flagging a bacteriophage with a detectable flag;

(b) combining said flagged bacteriophage with said sample and providing conditions sufficient to allow said flagged bacteriophage to bind to said microorganism to create a bacteriophage exposed sample;

(c) performing a target separation process, said separation process capable of separating said bacteriophage exposed sample into a target microorganism portion containing target mmicroorganisms present in said sample and an unbound flagged bacteriophage portion containing flagged bacteriophage that are not bound to said target microorganism; and

(d) assaying at least one of said portions to detect the presence or absence of said flag to determine the presence or absence of said target microorganism in said sample.

2. A method as in claim 1 wherein said microorganism is a bacterium and said assaying comprises detecting said flag as an indication of the presence of said target bacterium in said sample.

3. A method as in claim 1 wherein said assayed portion is said target microorganism portion.

4. A method as in claim 1 wherein said combining comprises applying said sample to a lateral flow strip.

5. A method as in claim 1 wherein said performing a target separation process comprises capturing said flagged and bound phage with a capture element specific to said target microorganism.

6. A method as in claim 1 wherein said flagging comprising attaching to said bacteriophage a detectable element selected from the group consisting of: gold particles, lectins, aptomers, immunoadhesion molecules, biotin molecules, polystyrene beads, latex beads, fluorophores and other color-based molecules.

7. A method as in claim 1 wherein said assaying comprises a method selected from the group consisting of: detecting color, detecting a shade, detecting fluorescence, detecting luminescence, immuno-detection, electrical detection and magnetic detection.

8. A method as in claim 1 wherein said assaying comprises detection with an instrument.

9. A method as in claim 1 wherein said assaying comprises determining the concentration of said target microorganism in said sample.

10. A method as in claim 1 wherein said performing a target separation process comprises washing said exposed sample.

11. A method as in claim 10 wherein said washing comprises centrifugation and decanting the supernatant.

12. A method as in claim 10 wherein said washing comprises filtering said bacteriophage exposed sample through a filter that allows unbound phage to pass through while retaining the bound phage.

13. A method as in claim 10 wherein said washing comprises rinsing with a wash solution.

14. A method as in claim 13 wherein said rinsing comprises rinsing with a low salt, neutral pH, or tris-based buffer.

15. A method as in claim 1 wherein said sample is a fluid and said combining comprises pouring said sample into a container containing said flagged bacteriophage.

16. A method as in claim 1 wherein said performing a target separation process comprises diffusion of said bacteriophage.

17. A method as in claim 1 wherein said combining comprises adding said sample to one of a plurality of chambers in a vial.

18. Apparatus (140) for detecting a target microorganism, said apparatus comprising: a substrate (164); a conjugate pad (143) on or in said substrate, said conjugate pad containing bacteriophage (70) conjugated with a detectable flag (70), said bacteriophage specific to said target microorganism; and an immobilization zone (146) on or in said substrate, said immobilization zone including an immobilization element (170) designed to immobilize said target microorganism.

19. Apparatus as in claim 18 wherein said immobilization zone comprises antibodies, aptamers, or bacteriophage.

20. Apparatus as in claim 18 wherein said flag comprises a colored element.

21. Apparatus as in claim 18 wherein said flag comprises a conductive element.

22. Apparatus as in claim 21 wherein said flag comprises gold.

23. Apparatus as in claim 18 wherein said flag comprises a magnetic element.

24. Apparatus as in claim 18 wherein said substrate comprises a lateral flow strip.

25. Apparatus as in claim 18 wherein said microorganism is a bacterium.

26. Apparatus as in claim 18 and further comprising an internal control zone

(148).

27. Apparatus (400, 600) for detecting a target microorganism (410, 610), said apparatus comprising a flagged bacteriophage (142) and a separation element (420, 620) that permits said flagged bacteriophage to pass but does not allow said target microorganism to pass.

28. Apparatus as in claim 27 wherein said separation element is selected from the group consisting of a filter and a membrane.

29. Apparatus as in claim 27 wherein said apparatus further comprises a vial (650) having two chambers (652, 654), with said separation element separating said two chambers.

30. A method of manufacturing a microbial test apparatus (140), said method comprising: providing a substrate (164) and a biological material (170) capable of attaching to a target microorganism; forming a line (146) of said biological material on said substrate; and cutting said substrate in a direction essentially perpendicular to said line to form said test substrate.

31. A method as in claim 30 and further comprising forming a sample pad and forming a supply (143) of bacteriophage conjugated with a detectable flag, said supply of bacteriophage being between said sample pad (141) and said biological material.

32. A method as in claim 30 wherein said substrate is a porous membrane.

33. A method as in claim 30 wherein said biological material is an antibody.

34. A method as in claim 30 wherein said providing comprises providing a first biological material and a second biological material, and said forming comprises forming a first line with said first biological material and a second line with said second biological material, with said first line and said second line being substantially parallel.