JP4578478B2 - Apparatus and method for detecting microorganisms using bacteriophage - Google Patents

Description

(Background of the Invention)
(1. Field of the Invention)
The present invention relates generally to the field of microbial detection, and more particularly to the detection of bacteria using bacteriophages.

(2. Description of problem)
Standard microbiological methods for the detection of microorganisms rely on substrate-based assays to check for the presence of specific bacterial pathogens. Robert H. Bordner, John A. et al. Winter and Pasquale Scarpino, Microbiological Methods For Monitoring, The Environment, EPA Report No. EPA-600 / 8-78-017, U.S. Pat. S. See Environmental Protection Agency, Cincinnati, Ohio, 45268, December 1978. These techniques are generally easy to implement, do not require expensive equipment or research equipment, and provide a high level of selectivity. However, the above method is slow. A barrier to substrate-based assays is that a pure culture of the target organism must first be grown or cultured, which can take 24 hours or more. This time constraint greatly limits the effectiveness of providing a rapid response to the presence of toxic strains of microorganisms.

  Molecular biology techniques are rapidly gaining acceptance as an alternative to standard microbiological testing. Serological methods are becoming widely used to evaluate matrix hosts for targeted microorganisms. David T.M. Kingsbury and Stanley Falkow, Rapid Detection And Identification of Infectious Agents, Academic Press, Inc. , New York, 1985 and G.C. M.M. Wyatt, H.C. A. Lee and M.C. R. A. See Morgan, Chapman & Hall, New York, 1992. These tests focus on using antibodies to first capture and then separate target organisms from other components in the complex organism mixture. Once isolated, the captured organic matter can be concentrated and detected by a variety of methods that do not require the culture of biological specimens. One such technique, called “Immunomagnetic Separation (IMS)”, is the immobilization of antibodies to microscopic magnetic or paramagnetic beads of a sphere and uses these beads to target microorganisms from a liquid medium. Including capturing. These beads are easily manipulated under the influence of a magnetic field that facilitates recovery and concentration of the target organism. Furthermore, because of their small size and shape, the beads can be uniformly dispersed in the sample, accelerating the interaction between the beads and the target. These preferred features reduce assay time, help streamline analysis procedures, and make them suitable for higher sample throughput and automation.

  The downstream detection method that has been used previously with IMS, ELISA (Kofitsyo S.Cudjoe, Therese Hagtvedt, and Richard Dainty, "Immunomagnetic Separation of Salmonella From Foods And Their Detection Using Immunomagnetic Particle", International Journal of Food Microbiology, 271 (995) 11-25), dot blot assays (Eystein Skjerve, Liv Marit Rorvik, and Orna Olswick, “Detection Of Listeria Monocytog. nes In Foods By Immunomagnetic Separation ", Applied and Environmental Microbiology, 11 May 1990, pp. 3478-3481), electrochemiluminescence (HaoYu and John G.Bruno," Immunomagnetic-Electrochemiluminescent Detection Of Escherichia coli 0157 and Salmonella typhimurium In Foods and Environmental Water Samples ", Applied and Environmental Microbiology, February 1996, pages 587-562), and flow site Tree (Barry H.Pyle, Susan C.Broadway, and Gordon A.McFeters, "Sensitive Detection of Escherichia coli 0157: H7 In Food and Water By Immunomagnetic Separation And Solid-Phase Laser Cytometry", Applied and Environmental Microbiology, 1999 years 5 Moon, pp. 1966-1972). Although these tests give satisfactory results, they are labor intensive to perform and give double reactions (yes / no) that are susceptible to false positive results due to cross-reactions with non-target analytes. Another method for identifying whole cell microorganisms is Matrix Assisted Laser Desorption / Ionization (MALDI) Time of Flight (TOF) Mass Spectrometry (MS) (Holland et al., 1996; van Barr, 2000; Madonna et al., 2000). Use.

  All of these new approaches can give results faster than conventional microbial methods. However, they cannot achieve the level of sensitivity achieved with substrate-based assays and are expensive and typically require skilled technicians than with classical substrate-based methods. Become.

  Another molecular biological technique that has received much attention in recent years is the polymerase chain reaction (PCR) method. PCR detection of specific microorganisms in a sample includes extraction of genetic material (RNA and / or DNA) in the sample, amplification of a target genetic sequence specific to the microorganism of interest, and detection of amplified genetic material. included. PCR technology provides high selectivity due to the uniqueness of the genetic material being detected, high sensitivity due to substantial amplification of the target genetic material, and rapid results due to the potentially rapid amplification process. However, PCR instruments and reagents are quite expensive and require highly skilled technicians to perform the tests. At present, PCR instruments do not give the expected sensitivity or specificity.

  Several attempts have been made to use bacteriophage infection and / or amplification to improve classical bacterial detection based on substrates. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replication. A bacteriophage (or phage) does this by attaching itself to the bacterium and injecting its genetic material into the bacterium to induce tens to thousands of times phage replication. Some bacteriophages, called lytic bacteriophages, release progeny phages into the environment so as to rupture the host bacteria and seek out other bacteria. The total incubation time for bacterial phage infection, phage replication (amplification) in bacteria, and release of progeny phage after lysis takes only 1 hour depending on the phage, bacteria and environmental conditions. Microbiologists have isolated and characterized more than 5,000 phage, including many that specifically target bacteria at the species or strain level. US Pat. Nos. 5,985,596 and 6,461,833 (Wilson) disclose such phage-based assays. This method involves infecting a sample that may contain bacteria of interest with a lytic phage. The free phage is then removed from the sample, the target bacterium is lysed, and the progeny phage is infected with a second bacterium, which has a shorter doubling time than the target bacterium. The prepared sample is cultured on a substrate. When plaques are formed, it can be seen that target bacteria are present in the original sample. This method can reduce the assay time of traditional substrate-based assays, although it still takes hours or days due to the culture incubation time required. Another problem with this method is that it can only be applied to detect bacteria in which there are non-specific phages that infect bacteria that double more rapidly than the target bacteria. The use of non-specific phage leads to the possibility of cross-reactivity with at least a second bacterium in the test sample. Therefore, this phage-based plaque assay is not rapid and is only applicable when appropriate non-specific phage are available, is prone to cross-reaction problems, and must be performed in laboratory facilities.

  The detection of other bacterial pathogens has completely abandoned the substrate-based Puark detection method. Many of these methods use bacteriophages genetically modified with a lux gene that is expressed only when the target bacterium is present in the sample and the modified phage infects. U.S. Pat. No. 4,861,709 (Ulitzur) is a typical example. Phages that specifically infect the target pathogen are modified to contain the lux gene. When a modified phage is added to a sample containing a target bacterium, the phage infects the bacterium, producing luciferase in the bacterium and causing luminescence. US Pat. No. 5,824,468 (Scherer et al.) Describes a similar method. In addition to luciferase production gene markers, Scherer et al. Describe genetic markers that are expressed as detectable proteins or nucleic acids. US Pat. No. 5,656,424 (Jurgensen et al.) Discloses a method for detecting mycobacteria using luciferase (or β-galactosidase) reporter phage. The patent also describes antibiotic susceptibility testing. US Pat. No. 6,300,061 (Jacobs Jr. et al.) Discloses mycobacterial detection using genetically modified phage that produces one of several reporter molecules including luciferase after bacterial infection. Yet another method for is described. US Pat. No. 6,555,312B1 (Nakayama) describes a method that utilizes a gene that produces a fluorescent protein marker rather than a luminescent one. All these methods take advantage of the potential advantages of phage amplification within infected bacteria. For each infected target bacterium in the sample, the marker gene is expressed many times during the generation of progeny phage. US Pat. No. 6,544,729 (Sayler et al.) Adds yet another amplification process. The phage DNA is modified to include the lux gene. Bioreporter cells are also modified to contain the lux gene. The genetically modified phage and bioreporter cells are added to the sample. When the phage infects the target bacterium, the target bacterium is induced to produce not only luciferase but also acylene homoserine lactone N- (3-oxohexanoyl) homoserine lactone (AHL). AHL finds a course in bioreporter cells and stimulates further light and further production of AHL, which finds a course in further bioreporter cells and produces more light. In this way, the light signal is amplified, triggered by the phage infection of the target bacteria. In principle, all of these methods that utilize genetically modified phage can detect 1) high selectivity to use selectively infecting phage and 2) marker gene products at the twelve level. High sensitivity and 3) Signals can be detected within 1 or 2 phage infection cycles, allowing rapid results compared to substrate based methods. These have two major drawbacks. First, it is expensive and difficult to implement because the appropriate phage must be genetically modified for each pathogen tested. Second, it often requires an instrument to detect the marker signal (light), which increases the cost of testing utilizing genetically modified phage.

  US Pat. No. 5,888,725 (Sanders) describes a method that utilizes unmodified highly specific lytic phages for infection of target bacteria in a sample. Phage-induced lysis releases specific nucleotides, such as ATP, detectable by known techniques from bacterial cells. If an increase in nucleotide concentration in the sample is detected after phage infection, it can be determined that the target bacterium is present in the sample. US Pat. No. 6,436,661 B1 (Adams et al.) Uses phage to infect and lyse target bacteria in a sample to release intracellular enzymes, which react with an immobilized enzyme substrate, A method for generating a detectable signal is described. These methods have the advantage of using unmodified phage, but do not derive any benefit from phage amplification. The concentration of marker (nucleotide or enzyme) detected is directly proportional to the concentration of target bacteria in the sample.

  US Pat. No. 5,498,525 (Rees et al.) Describes a pathogen detection method using unmodified phage and phage amplification to increase the detectable signal. This method claims that a high concentration of lytic phage is added to the sample. The sample is incubated for a time sufficient for the phage to infect the target bacteria in the sample. Before lysis occurs, the sample is processed to remove, destroy, or inactivate any free phage in the sample without affecting the progeny phage that replicate within the infected bacterium. If necessary, subsequent treatments are performed to neutralize any antiviral agents previously added to the sample. Progeny phage released by lysis is detected using a direct assay of the progeny phage or by using a genetically modified bioreporter bacterium that generates a signal indicating the presence of the progeny phage in the sample. . In either case, the measured signal is stronger as a result of phage amplification because it is proportional to the number of progeny phages, not the number of target bacteria in the sample. The main disadvantage of this method is that after killing, removing or inactivating free phage in the treated sample, the virucidal conditions must be reversed so that the progeny phage can survive the lysis. These additional processes complicate and expensive the assay using this method.

  There is a need for detection methods that combine the sensitivity, simplicity, and / or low cost of substrate-based assays with the rapid results provided by molecular biology diagnostic tests.

(Summary of the Invention)
The present invention solves the above problems as well as other problems of the prior art by providing a method and apparatus for detecting living microorganisms using the principle of phage amplification. In a preferred embodiment, a phage specific for the target microorganism is introduced into the sample. The amount of phage introduced is preferably an amount below the detection limit of the phage. If the target microorganism is present, the phage is infected and grows within the microorganism. Preferably, the microorganism is lysed either naturally as a result of phage growth, or by an active lysis process such as bacterial lysozyme if the microorganism is a bacterium. In a preferred embodiment, the phage is preferably dissociated by adding a bacteriophage dissociator. In another embodiment, prior to the detection process, the parent phage is labeled such that it can be physically removed or separated from the progeny phage, thereby increasing potential sensitivity and / or methods. Reduce total analysis time. The biological material associated with the phage or bacteriophage in the sample is then assayed. If any phage or biological material is detected, the presence of the target microorganism is verified. If no phage or biological material is detected, there will be no target microorganism. The entire incubation process consisting of infection, replication, and lysis takes only a few minutes. All of the foregoing embodiments can be used to determine antibiotic resistance or susceptibility of bacteria and other microorganisms. Bacteriophage or biological material can be detected in any suitable manner, such as using lateral flow strips, SILAS surfaces, or MALDI mass spectrometry.

  In a preferred embodiment, the present invention provides a method for detecting the presence or absence of microorganisms in a sample, the method combining a sample with a parental bacteriophage capable of infecting a target microorganism to produce a sample exposed to the bacteriophage. A dissociated bacteriophage that can be used in an assay as the bacteriophage infects a target microorganism and amplifies in the target microorganism to produce progeny bacteriophage for a sample exposed to the bacteriophage Providing conditions sufficient to produce the substance; and performing a test of the sample exposed to the bacteriophage to determine the presence or absence of the bacteriophage substance, which is an indicator of the presence or absence of the target microorganism in the sample. Including. In the above summary, it should be understood that a bacteriophage substance is a dissociated bacteriophage substance as well as related to a bacteriophage.

  In another preferred embodiment, the present invention provides a method for detecting the presence or absence of a microorganism in a sample, the method comprising: (a) exposing a sample to a bacteriophage in combination with a parental bacteriophage capable of infecting a target microorganism. And (b) for a sample exposed to a bacteriophage, the bacteriophage infects the target microorganism and amplifies in the target microorganism to detect a detectable amount of bacteriophage or Providing conditions sufficient to produce any of the biological material associated with the bacteriophage in the sample exposed to the bacteriophage; (c) actively lysing the microorganism; Bacteriophage or exposed to bacteriophage after assaying samples exposed to phage Detecting the presence or absence of a biological material associated with bacteriophage in sample, and a step of determining the presence or absence of a target microorganism. Preferably, the actively lysing step comprises adding microbial lysozyme to the sample exposed to the bacteriophage. Preferably, the actively lysing step comprises adding chloroform to the sample exposed to the bacteriophage, treating the sample exposed to the bacteriophage with acid, and physical processing of the sample exposed to the bacteriophage. A method selected from the group consisting of:

  Preferably, the process is realized using an apparatus and method in which the amplified phage induces a color change in the substrate. In a preferred embodiment, the present invention provides an apparatus for detecting a target microorganism, which is an immobilization zone on a substrate and immobilizing a bacteriophage or bacteriophage related biological material. An immobilization zone that contains an immobilizing agent for conversion to a bacteriophage or a bacteriophage-related biological material, thereby providing a bacteriophage or bacteriophage The presence of the relevant biological material causes a change in the color of the immobilization zone. Preferably, the immobilization zone comprises an antibody. Preferably the color modifier comprises colored beads. In another embodiment, the color modifier comprises a reactive agent and an enzyme that forms a precipitant upon reaction. In a preferred embodiment of the method, a sample exposed to a bacteriophage is applied to a substrate, and the bacteriophage or bacteriophage-related biological material is present in the sample exposed to the bacteriophage, at least a portion of its color To change.

  The present invention also provides a kit for determining the presence or absence of a target microorganism in a sample, the kit comprising a first container containing a bacteriophage capable of infecting a target microorganism and at least a portion of the color of the bacteriophage. And a substrate that changes when either bacteriophage or biological material associated with the bacteriophage is present in the sample exposed to the phage. Preferably, the kit further includes a second container containing a buffer solution. Preferably, the substrate consists of a lateral flow strip or a SILAS surface. Preferably, the first container includes a dropper designed to eject a predetermined size droplet.

  The present invention also provides a method of producing a test substrate of microorganisms, preferably bacteria, the method comprising providing a substrate and a biological material that can adhere to a bacteriophage or a biological material associated with a bacteriophage; Forming a line of biological material on the substrate; and cutting the substrate in a direction substantially perpendicular to the line. Preferably, the substrate is a porous membrane. Preferably the biological material is an antibody.

  It is an object of the present invention to provide a phage amplification method for detecting microorganisms that does not destroy, remove, neutralize or inactivate parental phage in a sample exposed to bacteriophage.

  An object of the present invention is to provide a highly specific method for detecting bacteria.

  It is a further object of the present invention to provide a broad spectrum bacteria detection method.

  Another object of the present invention is to provide a bacterial detection method that can be used to detect low concentrations of bacteria.

  Still another object of the present invention is to provide a method for detecting bacteria capable of detecting bacteria over a wide range of concentrations.

  It is a further object of the present invention to provide a bacterial detection method that provides rapid results compared to most existing detection methods.

  Still another object of the present invention is to provide a bacteria detection method that is less expensive than existing bacteria detection methods.

  Yet another object of the present invention is to provide a method for detecting bacteria that is easy to implement and does not require highly skilled technicians or complex equipment.

  It is a further object of the present invention to provide a method for detecting bacteria that can be performed in the field or at a nursing point.

  A further object of the present invention is to provide a method for detecting bacteria that rapidly doubles so that multiple bacteria are detected in a test sample.

  Yet another object of the present invention is to provide a bacterial detection method that uses detection of specific phage biomarkers as a surrogate for detecting target bacteria present in a sample.

  Yet another object of the present invention is to provide a bacterial detection method that utilizes a highly specific phage infection of a target bacterium as a means for specifically detecting the presence of the target bacterium in a sample.

  It is a further object of the present invention to provide a bacterial detection method that can be used to detect any bacteria in which the appropriate phage is present.

  It is a further object of the present invention to provide a method for bacterial detection that utilizes genetically unmodified phage.

  Yet another object of the present invention is to provide a method for detecting microorganisms utilizing genetically modified bacteriophages. For example, a bacteriophage may be inherited to enhance a desired property of the infection process, to overexpress a detectable biomarker, to express an enzyme, or to express a target on a capsid protein. Can be modified.

  It is a further object of the present invention to provide a bacterial detection method that uses phage amplification as a means of achieving high sensitivity.

  It is a further object of the present invention to provide a bacterial detection method for detecting phage by detecting specific biomarkers associated with phage, such as the capsid sheath of the phage.

  Yet another object of the present invention is to provide a bacterial detection method wherein the phage biomarker is a separate dissociated protein from the phage.

  A further object of the present invention is to provide a bacterial detection method wherein the phage biomarker is a phage nucleic acid.

  It is a further object of the present invention to provide a bacterial detection method that utilizes a second amplification process consisting of dissociating the phage prior to the detection process.

  Another object of the present invention is to provide a bacterial detection method that utilizes a labeled parent phage that is distinguishable from progeny phage produced by phage amplification.

  It is a further object of the present invention to provide a method for detecting bacteria using an antibody that binds to a phage biomarker that produces an antibody-phage complex.

  Yet another object of the present invention is to use existing immunoassay techniques to detect specific antibody-antigen binding events as a means for detecting antibody-phage complexes, and thereby in a sample. It is intended to provide a bacterial detection method for detecting the presence of target bacteria.

  Yet another object of the present invention is to provide a bacterial detection method that utilizes lateral flow strips to detect phage, thereby detecting the presence of target bacteria in a sample.

  Yet another object of the present invention is to provide a method for detecting antibiotic resistant strains of bacteria.

  The above summary is intended to illustrate some examples of the objects, features, and advantages of the present invention in order to facilitate the understanding of the present invention. In some embodiments of the present invention, only one of the above objectives may be realized, and in other examples, multiple of the above objectives may be realized. However, the above objectives are not all inclusive and are for illustrative purposes, and none of the above objectives may be realized in certain embodiments. For example, the methods and apparatus of the present invention can also be used to detect microorganisms other than bacteria, such as fungi, mycoplasma, protozoa, and other microorganisms. Therefore, if the term “bacteria” in the above object is replaced with the broader term “microorganism”, the object effective in the present invention is expressed. Numerous other features, objects and advantages of the present invention will become apparent from the following description when read in conjunction with the accompanying drawings.

Detailed Description of Preferred Embodiments
(1. Introduction)
The method of the present invention relies on the use of bacteriophage, or simply phage, to detect the presence of a target microorganism (microorganism) such as bacteria in a sample. In this disclosure, the terms bacteriophage and phage refer to bacteriophage, phage, mycobacteriophage (such as for TB and para-TB), mycophage (such as for fungi), mycoplasma phage or mycoplasma phage, and living It includes any other term that refers to a virus that can invade and use itself to replicate bacteria, fungi, mycoplasma, protozoa, and other microorganisms. Here, “microscopic” means that the maximum dimension is 1 millimeter or less. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replication. Phages do this by attaching themselves to bacteria, injecting their DNA into the bacteria, and inducing the bacteria to replicate the phage hundreds or thousands of times. Bacteriophages, called lytic bacteriophages, rupture host bacteria and release progeny phages into the environment to find their way to other bacteria. For bacterial lysis, the total incubation time for bacterial phage infection, phage growth or growth in bacteria is in any case tens to hours depending on the phage and bacteria and environmental conditions.

  The disclosed detection methods provide a combination of specificity, sensitivity, simplicity, speed, and / or cost that is superior to any currently known microorganism detection method. The method taught herein relies on using a bacteriophage to indirectly detect the presence of one or more target bacteria in a sample. A typical bacteriophage 70, in this case MS2-E. E. coli is shown in FIG. Structurally, bacteriophage 70 includes a protein shell or capsid 72, sometimes referred to as a head, that encloses viral nucleic acid 74, ie, DNA and / or RNA. The bacteriophage may further include an internal protein 75, a neck 76, a tail sheath 77, a tail fiber 78, and an end plate 79, and a pin 80. Capsid 72 is composed of repeated copies of one or more proteins. Referring to FIG. 10, when a phage 150 infects bacteria 152, the phage attaches itself to an appropriate site on the bacterial wall or membrane 151, and injects its nucleic acid 154 into the bacteria. Guide to be replicated to a thousand copies. This process is shown schematically in FIG. DNA expands into early mRNA S155 and early protein 156, some of which become membrane components along line 157, others utilize bacterial nucleases from host chromosome 159, along with DNA precursors along line 164 become. Others move along direction 170 and become head precursors that take up DNA along line 166. Membrane components develop along pathway 160 to form sheaths, endplates, and pins. Other proteins develop along pathway 172 and form tail fibers. Once the head is formed, it is released from the membrane 151 and joins the tail sheath along the path 174 and the tail sheath and head join the tail fiber at 176 to form the bacteriophage 70. Certain bacteriophages, called lytic bacteriophages, release progeny phages into the environment in order to destroy the host bacterium indicated at 180 and find a way to other bacteria. In the methods disclosed herein, lytic phages are typically used. However, non-lytic phages can also be used, particularly when non-lytic phages or bacteria can be activated to release the progeny phage or part of the progeny phage after the progeny phage has infected the host bacteria. .

The total cycle time for bacterial phage infection, phage growth or growth in bacteria in any case is tens of minutes to several hours depending on the phage and bacteria and environmental conditions. As an example, MS2 bacteriophage can infect a strain of Escherichia coli and produce 10,000 to 20,000 copies of itself within 40 minutes after attachment to a target cell. The capsid of MS2 phage contains 180 copies of the same protein. This is because each E. coli infected with MS2. This means that 1.8 × 10 ⁶ or more individual capsid proteins are produced for E. coli. The process of phage infection in which a large number of phage per infectious event, as well as more capsid proteins are produced, is called phage amplification.

  Microbiologists have isolated and characterized thousands of phage species, including specific phages for most human bacterial pathogens. Bacteriophage species may infect bacterial families, infect individual species, or infect specific strains. Table 1 lists some such phages and the bacteria that the phages infect.

本発明は、高度に特異的なファージ−細菌感染、ファージ増幅、および短いインキュベーション時間などのバクテリオファージの既存の特性を利用しており、標的細菌に高度に特異的で、非常に感度が高く、迅速で、実施が容易であり、および／または相当に経済的である細菌検出方法を編み出している。さらに、他のファージに基づく細菌検出方法とは違って、本願に記載する好ましい方法は、バイオレポータまたはインデューサ遺伝子を含むような遺伝的修飾を受けていないファージを利用している。このことにより、本発明を使用した特異的細菌試験の開発に伴う時間および費用が大幅に削減される。

The present invention takes advantage of the existing properties of bacteriophage such as highly specific phage-bacterial infection, phage amplification, and short incubation times, is highly specific to the target bacteria and is very sensitive, It has devised a bacterial detection method that is fast, easy to implement and / or quite economical. Furthermore, unlike other phage-based bacterial detection methods, the preferred methods described herein utilize phage that have not been genetically modified to include a bioreporter or inducer gene. This greatly reduces the time and expense associated with developing specific bacterial tests using the present invention.

(2. Detailed explanation)
FIG. 1 shows a first embodiment 10 of a method for detecting specific bacteria in a sample. In a first “phage addition” process 12, the parent bacteriophage 18 that will infect the target bacteria 14 is mixed with the original sample 11 of bacteria 14. In a preferred embodiment, bacteriophage, preferably in suspension or solution 16, is added to the original sample 11 of bacteria 14 at a predetermined concentration. Here, the term “original sample” refers to a sample before adding phage. The original sample / phage mixture is referred to herein as “test sample 24” or “sample 24 exposed to bacteriophage”. If the purpose of the method is to detect a specific bacterium at the species or strain level, then the corresponding specific phage is used in the method. For example, Y.M. Pestis. ΦA1122 phage can be used to specifically detect. On the other hand, in order to detect a wider range of bacteria in a sample, it is better to use a phage with lower specificity. Phage MS2 is not only enterococci but also many different E. coli. Since it infects E. coli species, it is very suitable for detecting fecal contamination in water.

  To detect multiple bacteria, one species of bacteriophage for each target bacterium is added to the original sample, giving a single sample containing phage associated with all target bacteria. For simplicity, the method is described below as applied to the detection of one bacterium. One skilled in the art will know how to perform each process of the method synchronously with one test sample using a unique bacteria / phage combination to detect each target bacterium. .

  The original sample 11 containing the target bacteria 14 is generally a liquid, but may be a solid or a powder. The original sample may be a mixture or suspension containing many different organic and inorganic compounds. The original sample may be pretreated in various ways to prepare for testing. For example, the original sample may be purified or filtered to remove unwanted components or to concentrate target bacteria. Alternatively, the cells may be cultured in a medium so as to cause incubation of the target bacteria or to make the target bacteria more easily viewable. The original sample may be in a relatively untreated state, such as a saliva, blood, or water sample. It will be apparent to those skilled in the art that the pre-test sample preparation may include any of a wide range of suitable processes, and the original sample may take a wide variety of forms.

  The phage itself may be added to various forms of samples. It may be added in a dry state. The phage may be mixed or suspended in the liquid reagent mixture. The phage may be suspended in a vial to which the original sample is added. The phage may take any other suitable form. The phage added to the original sample is referred to herein as the “parent phage”.

  Returning to FIG. 1, the test sample 24 is preferably incubated for a predetermined time. For this method, the test sample should preferably be in a condition that assists the phage infection of the target bacteria prior to the incubation process. This can be achieved by various methods known by those skilled in the art. For example, the parent phage may be mixed in a reagent that when added to the original sample results in a test sample that aids infection. Test samples may be prepared by various methods to establish conditions that aid in phage infection.

  An “incubation” process 20 is illustrated in FIG. The parent phage 18 infects the target bacterium 14 by attaching itself to the target cell wall (32), and injects viral nucleic acid to produce the infecting bacterium 23. Then, progeny phage replication 34 as shown in FIG. 10 proceeds in the host bacterium. If lytic phage is used, the host may rupture in the lysis process 36, releasing progeny phage 37 into the test sample, where it may infect other target bacteria. This incubation process may proceed during one or more infection, amplification, and lysis cycles. Assuming that the target bacteria are present in the original sample, the test sample will contain a large number of progeny phage for the individual bacteria infected during the incubation process.

  At the end of the “incubation” process, some of the target bacteria may not have been lysed. In such situations, many or all of the progeny phages are still retained in the host bacteria and this may not be directly detectable. To address this potential problem, at 21 the microbial lysozyme 22 for a particular microorganism is added to the test sample at 21 and the cell wall of essentially all individual microorganisms such as bacteria present in the test sample 24 at process 25. Is performed to release essentially all the progeny phage contained therein, and optional processes 21 and 25 “bacterial lysis” are performed as shown in FIG. The use of microbial, or more particularly bacterial lysozyme, can reduce the time required to perform the methods taught herein. This is because there is no need to wait for all of the target bacteria to lyse themselves. For phage that slowly lyse, this can make a substantial difference. For the purposes of the present invention, the term “lysozyme” refers to, but is not limited to, any material, device, or process that induces destruction of a microbial host to release progeny phage into a sample. , Including conventional chemical means such as lysozyme and chloroform, or acid treatment or physical processes such as the addition of osmotic pressure.

  Process 28 “Detection of phage” in the embodiment shown in FIG. 1 includes detecting biomarkers associated with the phage. When this biomarker is detected, this indirectly indicates the presence of the target bacteria in the original sample. For the embodiment of the method to be described, the parental and progeny phage added to the original sample are the same if produced during the incubation process. This means that there are phages during the detection process 28 that can raise the associated background signal even if the target bacteria are not present in the test sample. A way to solve this problem is to control the initial concentration of the parent phage in the test sample so that the background signal they give is undetectable in the detection process 28. Thus, if no target bacteria are present in the test sample, such as no phage amplification has occurred, no signal is detected at the end of the test. Higher concentrations of parental phage can be used only if the background signal can be distinguished from increased signal from the parental plus progeny phage. The lowest bacterial concentration at which the signal obtained in process 28 can be distinguished from the background signal represents the sensitivity limit of the method. A simple way to do this is to test against a control sample that is known to be free of bacteria. This can produce a control result that can be compared with the corresponding result from the test sample. If the test result from the test sample clearly shows a higher signal level than the control sample, it is found that the target bacteria are present.

  For the purposes of the present invention, any biomarker associated with the phage can be used as an indirect means of detecting the target bacteria in the original sample. This includes any portion of the phage shown in FIGS. For example, FIG. 11 shows a MALDI spectrum 200 representing the percentage of intensity with respect to mass for bacteriophage T4. The spectrum 200 shows a prominent peak 201 for the lysed holin protein, a prominent peak 204 for the head protein, a prominent peak 206 for the hoc external capsid protein, and a prominent peak 208 for fibrin. These large peaks indicate that any of these phage moieties can be used as biomarkers in the MALDI detection method discussed later. A very useful phage biomarker is phage capsid 72. The capsid usually contains many copies of one or more proteins, over 100 copies. Thus, there are a plurality of identical binding sites that can be used for detection purposes around each individual phage particle. This increases the potential signal level obtained from each phage particle contained in the test sample. The advantage of detecting phage or phage-related biomarkers over directly detecting biomarkers associated with lysed target bacteria is a dramatic increase in sensitivity. The phage concentration in the incubated test sample is much higher than the concentration of target bacteria in the target bacterial source due to phage amplification. Phage amplification can enhance the signal associated with each individual bacterium present in the original sample by an order of magnitude, eg, 10-fold. Thus, lower concentrations of bacteria can be detected with this method than other methods that do not use a phage amplification process.

  Any detection method or apparatus that detects any biomarker associated with the phage is sufficient for this method 28. Preferred methods utilize antibody binding events to generate a detectable signal, including ELISA, flow cytometry, Western blot, aptamer based assays, radioimmunoassay, immunofluorescence, and lateral flow immunochromatography (LFI) This is an immunoassay method. Other methods include matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), referred to herein as MALDI, and the use of a SILAS surface that changes color as a detection indicator. . One immunoassay, LFI, is discussed in detail below in connection with FIGS. 3, 5 and 19, and the SILAS color index method is discussed in detail below in connection with FIGS. 12-18, and MALDI. The method is discussed in detail below in connection with FIG. 11 and FIGS.

  FIG. 3 illustrates how an LFI for detecting the presence of phage in a test sample can be implemented using the lateral flow strip 40. A cross-sectional view of the lateral flow strip 40 is shown in FIG. The lateral flow strip 40 preferably includes a sample application pad 41, a conjugate pad 43, a substrate 64 on which a detection line 46 and an internal standard line 48 are formed, and an absorption pad 52, all of which are preferably It is mounted on a backing 62 that is plastic. The substrate 64 is preferably a porous mesh or film. This is created by forming lines 43, 46, and optionally lines 48, on a long sheet of the substrate, and cutting the substrate in a direction perpendicular to these lines to form a plurality of substrates 64. Each conjugate pad 43 includes colored beads 45 joined to a first antibody 44, each referred to herein as a first antibody forming a first antibody-bead conjugate. The first antibody 44 selectively binds to the phage 51 in the test sample. The detection line 46 and the control line 48 are both reagent lines, and each form an immobilization zone. That is, it contains materials that interact in an appropriate manner with bacteriophages or other biomarkers. In a preferred embodiment, the interaction is such that it immobilizes bacteriophages or other biomarkers. The detection line 46 includes an immobilized second antibody 47, which is perpendicular to the flow direction along the strip and has sufficient density to capture a significant portion of the phage in the flow. is doing. The second antibody 47 also specifically binds to the phage 51. The first antibody 44 and the second antibody 47 may or may not be the same. Either may be a polyclonal or monoclonal antibody. Optionally, strip 40 may include a second reagent line 48 that includes a third antibody 49. The third antibody 49 may or may not be the same as one or more of the first and second antibodies. The second reagent line 48 may serve as an internal standard zone for checking whether the assay functions properly.

  One or more drops of test sample 50 are added to the sample pad as shown in FIG. 3A. Test sample 50 preferably includes parental phage as well as progeny phage if the target is present in the original original sample. The test sample flows along the lateral flow strip 40 toward the absorbent pad 52 at the opposite end of the strip. As the phage particles flow toward the membrane along the conjugate pad, they incorporate one or more of the first antibody-bead conjugates 42 that form the phage-bead complex 54, as shown in FIG. 3B. As the phage-bead complex travels over the row 46 of the second antibody 47, the immobilized and concentrated first antibody-bead-phage-second antibody complex 58, as shown in FIG. 3C. Form. When sufficient phage-bead complex is bound to the immobilized second antibody array 46, the colored line 59 becomes visible to the naked eye. Visible line 59 indicates that the target bacterium was present in the original sample. If no line is formed, the target bacteria were not present in the original sample, or were present at such a low concentration that they cannot be detected by the lateral flow strip 40. To obtain the reliability of this test, the concentration of parental phage added to the original sample should be low enough so that the parental phage alone does not have an amount that can form a visible line on the lateral flow strip. is there. Antibody-bead conjugate 45 is a toning agent designed to interact with bacteriophages or biological materials associated with bacteriophages. When antibody-bead conjugate 45 is immobilized in immobilization zone 46, it causes a color change in the immobilization zone.

  FIG. 6 shows a second embodiment 90 of a method for detecting target bacteria according to the present invention, which method 90 has improved sensitivity. Processes 12, 20 consisting of "addition of phage", "incubation" and "bacterial lysis", and optional process 21, are the same as the corresponding processes described with respect to FIG. After optional process 21, the test sample is rich in phage particles if present in the target bacteria in the original sample.

  As shown in FIG. 6, the process 94, “phage dissociation” of the second embodiment 90 includes adding a phage dissociator 92 to the test sample. The phage dissociator 92 breaks down the phage particles into their components 97, including individual capsid proteins and viral nucleic acids. Examples of phage dissociating agents include acid treatment, urea, denaturing agents and enzymes. Any suitable phage dissociator can be used. In this process, dissociated bacteriophage material 97 is produced.

  Process 99 “Detection of phage subcomponents” in the embodiment shown in FIG. 6 includes detecting biomarkers, ie, dissociated bacteriophage material 97 associated with dissociated phage subcomponents. With respect to the above discussion, it should be understood that the bacteriophage material can be both dissociated bacteriophage material and bound to the bacteriophage. That is, the term “dissociated bacteriophage substance” refers to something that is no longer part of the total bacteriophage, and the term “associated with bacteriophage” means that the substance is part of the bacteriophage at some point. It was produced during the bacteriophage replication process. By using a phage dissociator in process 94, there are abundant individual capsid proteins 97 detectable in process 99. Similar to the first embodiment, they can be detected using established antigen-antibody based immunoassay techniques. In addition, exposed viral genetic material can be detected using PCR, genetic probe biosensors, photoaptamers, molecular beacons, or other established techniques including gel electrophoresis. Any suitable phage biomarker detection method or apparatus can be used.

  By keeping the concentration of the parent phage of the test sample below the background detection limit, a simple method can be made of adding phage to the original sample, incubating, and detecting the phage biomarker. However, there are also potential drawbacks. A potentially low concentration of parental phage creates conditions where the ratio of parental phage to target bacteria in the test sample is less than 1, ie, a low multiplicity of infection (MOI). In order to ensure that all target bacteria in the test sample are infected with a high probability, the incubation time in process 20 can be increased, for example, equivalent to two or more cycles of infection and lysis. In this way, test simplicity is compensated for by potentially long test times. This potential limitation can be overcome if the signal associated with the parent phage can be eliminated or significantly reduced so that higher concentrations of the parent phage can be utilized (MOI> 5). The above limitations can also be overcome by using a capsid protein as a biomarker to enhance the signal from progeny phage or using genetically enhanced phage, both of which are described herein. Consider in detail.

  FIG. 7 shows a third embodiment 100 of the present invention for detecting target bacteria in a sample, in which a faster result can be achieved. In this embodiment of the present application, the parent phage 102 to be mixed with the original sample is labeled and indicated by the tag 104 and can be later removed from the test sample and isolated from the portion of the test sample that detects bacteriophage Otherwise, or neutralize prior to analysis so that the first untagged progeny phage contributes to the detected signal. For example, in one embodiment, biotinylated phage is used as the parent phage. Biotinylated bacteriophages are strongly attracted to streptavidin. This strong affinity was used to later isolate the labeled parental phage from the test sample, as discussed below. The labeled parental phage can also be attached to a physical substrate, for example, by coating the probe or network structure with the parental bacteriophage, or by chemically binding the phage to the substrate. The labeled bacteriophage can be separated from the progeny bacteriophage by removing the substrate from the test sample or by detecting the bacteriophage in a portion of the test sample separated from the substrate.

  In the processes 105, 107 and 108, “addition of phage”, “incubation” and “bacterial lysis” of the present embodiment, the solution of the parent phage 103 added to the original sample in the process 105 is labeled with the labeled phage 102, respectively. 1 and 6 except that the sample 109 contained and thus exposed to the bacteriophage contains both labeled phage 102 outside the bacteria and unlabeled progeny phage 106 inside the bacteria. , 20 and 21. Therefore, the lysis solution 112 will contain both labeled and unlabeled phage. In process 114, “extraction of labeled phage”, the labeled parent phage extracts the phage from the test sample or substantially removes it, or isolates the parent phage from the progeny phage and signals that are analyzed. It is separated from the progeny bacteriophage by making no contribution to. If the labeled parent phage is added to the physical substrate when added to the original sample in process 105, the substrate and associated parent phage can be physically separated from the test sample in process 114. preferable. Biotinylated phage that have not been added to the physical substrate can also be rapidly separated or removed from the test sample. In one embodiment, streptavidin-coated magnetic beads were added to the test sample, where the biotinylated parental phage was rapidly recovered. The magnet is then used to separate and remove from the test sample along with the parental phage to which the magnetic beads are bound. See the magnetic extraction discussion associated with FIG. 28 below. Similarly, in another embodiment, a streptavidin-coated mesh is agitated through the test sample to recover substantially all of the biotinylated parental phage from the test sample. Other physical substrates or probes other than the net may be used. In another embodiment, a lateral flow device was used. The portion 66 (FIG. 4) of the mesh substrate 64 in front of the antibody strip 46 is impregnated with streptavidin to coat the mesh transition. The streptavidin-coated screen is collected and the labeled parental phage is immobilized by binding the parental phage to portion 66 before reaching the antibody strip 46. Progeny phage did not bind to streptavidin and therefore flowed down the strip freely and was detected visually. Similarly, other parts of the lateral flow device can be coated or impregnated with streptavidin, such as a sample pad 41 to which a test sample is dropped.

  The methods described herein are not limited to the above example of labeling the parent phage and then removing it from or separating within the test sample.

  Process 116 of the embodiment shown in FIG. 7, “Detection of phage” is for analyzing a test sample to detect biomarkers associated with progeny phage as surrogate markers for target bacteria present in the original sample. Is. The detection means used in this embodiment is the same as described with respect to processes 28 and 29 of embodiments 10 and 90 illustrated in FIGS. 1 and 6, respectively. As with the previous embodiment, any suitable detection method or apparatus can be used.

  FIG. 8 shows a fourth embodiment 120 of a method for measuring target bacteria in a sample, in which more rapid results can be achieved and sensitivity is improved. Embodiment 120 is a combination of the methods taught in embodiments 90 and 100. Processes 105, 107, 108 and 114 are taught in embodiment 100 and illustrated in FIG. 7, ie “addition of phage”, “incubation”, optionally “bacterial lysis” and “detection of labeled phage”. Are the same. Specifically, embodiment 120 is a combination of labeled parent phage in process 105 and parent phage removal or separation in process 114.

In process 121, “Phage dissociation”, a phage dissociator 122 is added to the test sample 124 as taught in process 94 of embodiment 90 and illustrated in FIG. In a preferred embodiment, the labeled parent phage is physically removed from the test sample in process 114, rather than simply separated so that it is not exposed to the phage dissociator in process 121. In this way, test sample 124 will contain only progeny phage and dissociated test sample 126 will contain biomarker material such as capsid protein 128 from only the progeny phage. In this way, amplification associated with dissociation of phage capsid protein 128, combined with phage amplification of process 107, greatly increases total amplification. For example, if the phage amplification process gives 1000 amplifications per bacterium and the phage has 100 copies of a particular capsid protein, combinatorial amplification is 10 ³ × 10 ² per target bacterium infected in the test sample or 10 ⁵ If the parent phage is not removed, the total amplification will be only the phage amplification occurring in process 107, ie 10 ³ , which means that the amplification caused by phage dissociation occurs for both parent and progeny phages, This is to cancel the second amplification process.

  The “detect phage subcomponent” process 130 of embodiment 120 shown in FIG. 6 is preferably identical to any of the processes 28, 99 and 116 of the previous embodiment.

  FIG. 9 illustrates a method 140 in which any embodiment of the present invention can be used to detect a target bacterium and, if present, determine whether it is resistant to one or more antibiotics. Show. The sample 142 that may contain the target bacteria is divided into a first sample A indicated by 144 and a second sample B indicated by 145. First antibiotic 146 is added to sample B, where the target bacteria in sample B are killed if they are not resistant to the first antibiotic. Samples A and B are analyzed at 148 and 149 to detect the presence of their respective live target bacteria, giving results A and B. Any method taught in the present invention can be used for these analyses. A positive result A indicates that the target bacteria are present in the original sample. If the result B is also positive, it indicates that the target bacterium is resistant to the first antibiotic. On the other hand, if the result B is negative, it indicates that the target bacterium is not resistant to the first antibiotic. In order to simultaneously screen for antibiotic resistance to any of a range of antibiotics, sample B can be foamed with all antibiotics of interest prior to analysis against the target bacteria. If the target bacteria are detected in both the pure sample and the sample treated with antibiotics, it indicates that the target bacteria in the sample are resistant to one of the added antibiotics. This process can also be used to determine the susceptibility of bacterial antibiotics or other decontamination equipment. It can also be used to determine if the bacterial decontamination process was successful. By dividing the sample into a control part and a test part, bacteriological methods and the effectiveness of the substance can be examined. One skilled in the art will appreciate that the process of the present invention can be used in almost all cases where it is desirable to determine whether live bacteria are present.

  FIGS. 12-18 illustrate another embodiment of the detection process 28, 99, 116 and 130. This process uses a SILAS surface 220. The SILAS surface 220 consists of a semiconductor or insulator wafer 221 that may have a coating 222 optionally covered with an additional polymer 224. As is well known in the art, SILAS surfaces are designed to reflect certain light wavelengths and attenuate others by interference. These surfaces generate a visible signal by direct interaction of light with the film formed on the surface. A thin film is a biofilm that includes an optical coating and / or is created by the binding of specific target molecules to a surface. A positive result is usually seen as a color change from gold to purple because the optical path of light is extended by the biomass accumulated on the surface. The thickness and refractive index of the film determine the individual colors and shading that are observed. In general, the wavelength of light reflected from the surface having the same phase as the incident light is added or received by constructive interference so that it can be visually recognized. Wavelengths reflected from surfaces that are out of phase with the incident light are attenuated by destructive interference and do not float from the film. Preferably, the wafer 221 is made of silicon, the optical coating is made of silicon nitride, and the addition polymer is made of a hydrophobic polymer. FIGS. 13-16 utilize a single, greatly enlarged structure showing a number of substantially uniformly distributed antibody / phage / antibody conjugates 231 on the surface of addition polymer 224, FIG. 5 shows how the SILAS surface is used to indicate the presence of phage markers. In FIG. 13, a first antibody 228 specific for a phage marker such as a capsid protein is added to an addition polymer, leaving the surface 225 of the polymer as an immobilization zone. A sample solution is brought into contact with surface 224. If the designated phage biomarker 230 is present, the marker is added to the first antibody 228 as shown in FIG. In FIG. 15, if a biomarker is present, the second detection antibody 232 is brought into contact with the surface 224 and added to the biomarker 230. The second antibody 232 is labeled with a reaction reagent such as horseradish peroxidase (HRP) or alkaline phosphatase. Then, an enzyme such as 3,3 ′, 5,5 ′ tetramethylbenzidine (TMB) is applied to the surface (236), and the enzyme reacts with HRP to form a precipitate 238, which forms the coating layer 240 (FIG. 16). ) To change the color of the surface. As shown in FIG. 16, the light 242 impinging on the surface 245 is variously reflected as indicated by 244 and 246 depending on whether or not the coating layer 240 is present. In this way, the combination of the second antibody 232 labeled with the reaction reagent, the enzyme and the optical coating 222 to the light modulating agent 235 that interacts with the bacteriophage or biological material associated with the bacteriophage, ie bacteriophage or The presence of biological material associated with the bacteriophage causes a color change in the immobilization zone 225.

  FIGS. 17 and 18 show the difference in reflectance between the SILAS surface without the coating layer 240 for negative results (FIG. 17) and the SILAS surface with the coating layer 240 for positive results (FIG. 18). Is. The reflectivity was higher at orange to red wavelengths for negative results and resulted in gold, and for positive results was higher at deep blue wavelengths and resulted in purple. The SILAS surface is available from Thermo Electron Corporation, 81 Wyman Street, Waltham, MA 02454-9046. For a description of the SILAS process, see the Thermo Electron Corporation website on the Internet, http: // www. thermo. com. Etc.

  FIG. 19 shows an exemplary test kit 254 for detecting microorganisms, along with typical instructions for using the test kit. See also FIG. Test kit 254 preferably includes a container 256 of buffer solution 258, a reaction container 260, one or more detection elements 266 (FIG. 20) enclosed in a protective case 263, and instructions 270 for using the kit. And a container 272 for holding the kit parts described above. The protective case 263 may include a control detection element 276 that indicates the expected result 267 in the absence of bacteria. For example, if the detection element is a lateral flow strip, the control detection element may be the same lateral flow strip coated with a bacteriophage control sample free of any bacteria. The reaction vessel 260 includes a vessel body 267 and a vessel lid 264. Preferably, the reaction vessel body is a bottle 267 and the reaction vessel lid is a bottle cap 264. Reaction vessel 260 contains phage 268. The phage 268 is preferably composed of a predetermined amount of phage added to the inner wall 269 of the reaction vessel main body 267. The cap 264 is preferably a threaded cap having an internal thread 262 that threadably engages the thread on the top of the bottle 267. The cap 264 preferably includes a dispenser 265, which is preferably a drip head designed to eject a predetermined size drop. In this embodiment, the sensing element 266 is a lateral flow strip 266, but may be a SILAS surface element as described with respect to FIGS. The flow strip 266 includes a sample pad 274 (FIG. 20) and a detection window 278. Preferably, the container 272 comprises a plastic bag 272 that serves the dual role of holding the test kit parts and a convenient disposal container after completion of the test.

  FIG. 20 shows an exemplary set of instructions 270 for using the test kit 254. The instructions 270 preferably consist of a sheet of paper on which letters and diagrams indicating the test procedure are printed. Instruction 270 also describes an exemplary method 280 for detecting microorganisms. Method 280 includes processes 281, 282, 284 and 286. In the first process 281, the reaction bottle 267 is opened by removing the cap 264 and the drip head 265 and a 5 milliliter (mL) sample is added. Then buffer solution 258 is added. In process 282, the dripping head 265 and the cap 264 on the bottle 267 are returned and the capped reaction vessel 260 is preferably stirred for a predetermined time, such as 1 minute, and then preferably for a predetermined amount of time, such as 1 hour. Incubate the solution by allowing to stand. The cap 264 is removed and a predetermined amount of the incubated sample is released onto the sample pad 278. The user then waits for a predetermined amount of time, for example 3 minutes. In process 286, the user looks through the detection window 278 to see the results. As discussed above, if the sample contains bacteria identified by the test kit, a first line 288 of the first color such as blue appears. If the first color line does not appear, the test result is negative. Optionally, it is preferred that a second line 290 of the second color appears to indicate that the test is valid. The first line 288 corresponds to the reagent line 46 of FIG. 3, and the second line 290 corresponds to the internal standard zone 48 of FIG. Control detection element 276 (FIG. 19) may be used to compare first line 288 with control line 277 to determine whether the test is positive or negative. For example, if the first line 288 is clearly deeper blue than the control line 277, a positive result is known.

  FIG. 21 illustrates an exemplary assay 300 according to the present invention using a bacteriophage 302 that has been genetically modified to enhance the desired properties of the infection process. As used herein, “genetically modified bacteriophage” includes both bacteriophages whose DNA has been modified or manipulated in some way and bacteriophages that have been selectively propagated to enhance certain properties. Desired properties include burst volume, burst time, and infectivity. The burst volume is the amount of phage that replicates, the burst time is the time it takes for the phage to rupture the target bacteria, and the infectivity is the effectiveness of the phage in the infected bacteria, i.e. a given amount of Percentage of available bacteria infected by phage. The phage may be genetically modified to enhance one or more of the desired properties and / or other desired properties. As shown in FIG. 21, a genetically modified phage 302 is used to detect microorganisms as in the case of unmodified phage. That is, an amount, preferably less than the limit of detection, of genetically modified phage 302 is added to a sample of host microorganism 304 (303), infected and incubated (305), generating phage progeny (306); This is detected (307). Since parental phage 302 has been genetically modified to enhance the characteristics of the infection process, detection can be made faster, more sensitive, and / or more reliable.

  FIG. 22 shows an exemplary assay 310 according to the present invention using a bacteriophage 312 genetically modified to overexpress a detectable biomarker such as a protein. As shown in FIG. 22, a genetically modified phage 312 is used to detect microorganisms as in the case of an unmodified phage. That is, an amount, preferably less than the limit of detection, of genetically modified phage 312 is added to a sample of host microorganism 314 (313), infected and incubated (315) to generate phage progeny (316); This is detected (317). Since parental phage 302 is genetically modified to overexpress a detectable biomarker, this biomarker is more easily detected, and thus detection proceeds faster and more sensitively, ie , Lower levels of bacteria can be detected. Alternatively, the amount of parent phage used can be reduced.

  FIG. 23 shows an exemplary assay 331 according to the present invention using a bacteriophage genetically modified to express an enzyme. As shown in FIG. 23, an amount, preferably less than the limit of detection, of genetically modified phage 322 is added to a sample of host microorganism 324 (323), infected, incubated (325), and the enzyme is generated. (326). Bacteria may or may not be lysed. Substrate 328 is added (327), which reacts with the enzyme (329) to provide enzyme product 330 or other enzymatic action, which is detected. This genetic modification can provide an alternative detection method that can be performed more easily, can proceed more rapidly and / or with high sensitivity, or can reduce the amount of parental phage used.

  FIG. 24 shows an exemplary assay according to the present invention using a bacteriophage genetically modified to express a target on a capsid protein. As shown in FIG. 24, ie, an amount, preferably less than the limit of detection, of genetically modified phage 332 is added to a sample of host microorganism 334 (333), infected, incubated (355), and capsid protein A phage progeny containing the biomarker 336 on the surface of 335 is generated (338). Since the biomarker is outside the capsid, it can be detected more easily than other markers and / or can be detected without a phage dissociation process, and the detection speed can be improved.

(3. Example)
(A. Lateral flow example)
In the following examples, E.E. in the process of FIG. MS2 phage is used for detection of E. coli. Lateral flow strips as shown in FIGS. 3 and 4 are prepared using a polyclonal antibody that specifically binds to MS2 phage.

Determination of MS2 detection limit for lateral flow strips-Bacteriophage MS2 (ATCC 15597-B1) was used for infection with E. coli on confluent plates. E. coli (ATCC 15597) cells. The concentration of live MS2 from this preparation was 2 × 10 ⁷ pfu / mL by plaque assay. This MS2 stock was serially diluted to give a range of 1 × 10 ⁷ pfu / mL to 1 × 10 ⁵ pfu / mL. MS2 was detected using a lateral flow strip. The results are shown in Table 2.

線強度は、試料をラテラルフローストリップに流してから１５分後に目視で判定した。線強度は、最大線強度を示す「＋＋」から、線を検出できない「−」までの尺度範囲上でランク付けした。「＋／−」は、かろうじて検出可能な線を示す。このアッセイの結果は、上記試験のために調製したラテラルフローストリップ上でのＭＳ２の検出限界が、１×１０^６ｐｆｕ／ｍＬであることを示している。

The line strength was visually determined 15 minutes after flowing the sample through the lateral flow strip. The line intensity was ranked on a scale range from “++” indicating the maximum line intensity to “−” where no line was detected. “+/−” indicates a barely detectable line. The results of this assay indicate that the detection limit of MS2 on the lateral flow strip prepared for the above test is 1 × 10 ⁶ pfu / mL.

E. E. coli detection limits and determination of total test time-E. coli from saturated cultures E. coli (ATCC 15597) is diluted to a concentration of 1 × 10 ⁸ , 1 × 10 ⁷ , 1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ , 1 × 10 ³ , and 1 × 10 ² cells / mL. An original sample was prepared. MS2 was added to each original sample to give a test sample with an MS2 concentration of 1 × 10 ⁶ pfu / mL. Test samples were incubated at 37 ° C for 1 to 5 hours. After incubation, the test samples were diluted 10: 1 so that the parent MS2 concentration was 1 × 10 ⁵ pfu / mL below the established detection limit. The test sample was then analyzed using a lateral flow strip. The results are shown in Table 3.

これらの結果は、開示した発明を用いて、４時間後に１００個のＥ．ｃｏｌｉ細胞を検出できたことを示す。より高いレベルのＥ．ｃｏｌｉ（１×１０^５細胞以上）が１時間後に検出可能である。

These results show that 100 E. coli after 4 hours using the disclosed invention. It shows that E. coli cells could be detected. A higher level of E. coli. E. coli (1 × 10 ⁵ cells or more) can be detected after 1 hour.

  Similarly, the lateral flow strip process described above uses PRD-1 bacteriophage with Salmonella choleraesu, gamma phage with Bacillus anthracis, and B-30 bacteriophage. It was successfully used to detect Group 8 Streptococcus.

(B. SILAS surface embodiment)
Methods: Coated surfaces and HRP-conjugated antibodies were prepared using standard methods developed in Thermo Electron Corporation, 81 Wyman Street, Waltham, MA 02454-9046. Briefly, the surface was coated in a solution of HEPES buffer (pH 7.8) containing 4 ug / ml rabbit anti-MS2 antibody for 48 hours. After coating, the wafer was washed, overcoated with a sugar: protein preservative, and separated into 7 mm square chips.

  Conjugation proceeded according to Nakae's method Thermo Electron modification. Sodium peroxide was used to activate the HRP to introduce the anhydride onto the carbohydrate portion of the protein. Activated HRP and rabbit anti-MS2 antibody were mixed and incubated. The conjugate was stabilized by adding sodium borohydride to reduce Sciff base.

  A test was conducted to determine the detectability of a given MS2. The first form included both synchronous and continuous forms. The synchronous format consisted of mixing the sample and conjugate (diluted 1: 100 in conjugate dilution) and adding the sample to the surface of the coated OIA chip. Following incubation, the surface was washed and dried, and then an enzyme substrate (TMB) was added. The first and second incubations were equilibrated for 5 or 10 minutes. The continuous assay was performed in the same manner as the synchronous format except that the sample and conjugate were not mixed and added individually to the chip. The incubation step was divided into washing and blotting steps. The continuous assay used a 10 minute incubation for all steps.

Results: The non-optimized method described here could clearly detect MS2 samples at 10 ⁷ , but the results were mixed for 10 ⁴ . The color change in all cases was from deep gold orange to deep purple. Using the simultaneous format, weak and barely visible results were detected in undiluted samples using two 10 minute incubations. Using a continuous method, the signal detected was stronger at 10 ⁷ . Three 10 minute incubations revealed a 10 ⁷ signal and a 10 ⁴ signal became visible, but very weak. Serial dilutions are 10 ⁷ samples was performed by diluting with physiological saline using a 2-fold dilution. Positive results were detected up to a 1:16 dilution of 10 ⁷ samples. This is approximately equal to 6.25 × 10 ⁵ .

(C. Antibiotic resistance)
In this example, the minimum inhibitory concentration (MIC) of antibiotics in Staphylococcus aureus (S. aureus) was rapidly determined by bacteriophage amplification using MALDI-MS. The MIC is The lowest antibiotic concentration that inhibits the growth of a particular strain of Aureus. If the strain is sensitive to the assay concentration of antibiotic, the bacteriophage biomarker signal will not be detected by MALDI-MS due to antibiotic inhibition and thus suppression of phage amplification. On the other hand, if a phage biomarker signal is detected, it means that MIC has not been achieved and antibiotics are not effective. Streptomycin and tetracycline were selected to determine the MIC for this study.

  S. Aureus (ATCC 27709, Manassas VA) was cultured for 24 hours in BHI broth in the presence and absence of antibiotics (streptomycin and tetracycline). Phage 187 and S. cerevisiae. After aureus samples were diluted below the detection limit of MALDI, phages were infected into the host and bacteriophage amplification was performed until the cells burst in the host. The sample was then cleaned and concentrated for analysis. Samples were plated for analysis on a hydrophobic target plate with a 15 mg / mL ferlasan matrix mixed in a solution of formic acid, acetonitrile and HPLC grade water using the dried drop method. Mass spectra in linear mode were obtained using a MALDI-TOF-MS PerSeptive Biosystems Voyager-DE STR Biospectrometry Workstation (Framingham, Mass.).

  S. in antibiotics By culturing aureus, it can be seen whether the antibiotic concentration is sufficient to destroy the bacterial cells. If the bacterial cells are alive, they are not affected by the constituents, indicating that phage amplification occurs when the cells are infected. The result is that protein markers for bacteriophages are found in the mass spectrum. On the other hand, if no biomarker is found in the mass spectrum, it indicates that the minimum inhibitory concentration has been reached or exceeded. In conclusion, the cells are destroyed and therefore no bacteriophage amplification can occur.

Prior to analysis on MALDI, the sample was filtered and concentrated using semi-purification techniques. Samples were spin filtered using a 100 kDa cutoff because the instrument was intolerant to salt. Mass spectrum from phage 187 showed a clearly distinguishable protein biomarker at 15,245 Da. Phage 187 was also purified by ultracentrifugation and a cesium chloride gradient, confirming that biomarkers were identified by semi-purification filtration techniques. MALDI MS limits of detection were established for both bacteria and phage, which were 10 ⁶ cells / mL and 10 ⁸ phage / mL, respectively. During the experiment, the bacterial and phage concentrations were kept below the detection limit of MALDI to confirm that the signal was derived from amplified phage. When the antibiotic concentration was low, protein peaks from the phage were present in the MALDI spectrum, indicating that bacterial growth and phage replication were still occurring. At higher concentrations, the spectrum lacked protein peaks, indicating that the MIC was reached or exceeded.

  A feature of a preferred embodiment of the present invention is that the parental phage is not destroyed, removed, neutralized or inactivated in the sample exposed to the bacteriophage. In conventional methods, extracellular bacteriophages, ie bacteriophages outside bacteria or other infected microorganisms, are destroyed, removed, neutralized or inactivated at some point. This is not necessary with the present invention. In particular, it is preferred not to destroy, neutralize or inactivate extracellular bacteriophages by the addition of agents that kill bacteriophages. This is because it unnecessarily complicates the method and can affect offspring bacteriophages if the drug is not removed or neutralized.

  A microorganism detection method has been described that is specific to the selected organism, sensitive, simple, rapid, and / or economical and has a number of new features. The present invention can be used in a wide range of applications including human clinical diagnosis, veterinary diagnosis, food pathogen detection, environmental testing and biological war detection. The specific embodiments illustrated in the drawings and described herein are for purposes of illustration and should not be construed as limiting the invention. The invention is set forth in the following claims. Moreover, those skilled in the art will be able to create many uses and variations of the specific embodiments described without departing from the concepts of the present invention. Equivalent structures and processes may be substituted for the various structures and processes described, and the sub-processes of the methods of the invention may be performed in a different order in some examples, or may include a variety of different materials and processes. Elements may be used.

FIG. 1 shows a first embodiment of the invention in which phage is added to a sample to give an initial concentration below the detection limit. FIG. 2 illustrates the incubation process of phage infection, amplification, and cell lysis. 3A, 3B, and 3C illustrate the use of a lateral flow device to detect phage in the test sample. 4 is a cross-sectional side view of the flow device of FIG. 3A. FIG. 5 is an explanatory diagram of bacteriophage. FIG. 6 shows a second embodiment of the invention in which phage is added to the sample to give an initial concentration below the limit of detection, where the phage detects a biomarker that is a small component of the phage Is dissociated. FIG. 7 shows a third embodiment in which labeled phage is added to the sample. FIG. 8 shows a fourth embodiment of the invention in which the labeled parent phage is added to the sample and the progeny phage is dissociated so that a biomarker that is a small phage component is detected. FIG. 9 shows detection of antibiotic resistant bacteria using the present invention. FIG. 10 shows the phage amplification process. FIG. 11 is a MALDI spectrum showing mass versus intensity for bacteriophage T4 showing several possible biomarkers. FIG. 12 shows a phage-based assay process using a SILAS surface. FIG. 13 shows a phage-based assay process using a SILAS surface. FIG. 14 shows a phage-based assay process using a SILAS surface. FIG. 15 shows a phage-based assay process using a SILAS surface. FIG. 16 shows a phage-based assay process using a SILAS surface. FIG. 17 shows the negative results from the assays of FIGS. FIG. 18 shows the positive results from the assays of FIGS. FIG. 19 shows a test kit according to the present invention. FIG. 20 shows exemplary guidance for using the test kit of FIG. 19 and describes samples of the test kit. FIG. 21 shows an exemplary assay according to the present invention using bacteriophage genetically modified to enhance the desired properties of the infection process. FIG. 22 shows an exemplary assay according to the present invention using a bacteriophage genetically modified to overexpress a detectable biomarker. FIG. 23 shows an exemplary assay according to the present invention using a bacteriophage genetically modified to express an enzyme. FIG. 24 shows an exemplary assay according to the present invention utilizing a bacteriophage genetically modified to express a target on a capsid protein.

Claims (10)

A method for determining the presence or absence of a target bacterium (14) in a sample to be tested, the method comprising:
Parent bacteriophage capable of infecting said target bacteria (18) together with the sample (12), a step of preparing a sample (16) that is exposures bacteriophage; and,
The exposures sample to the bacteriophage to infect the bacteriophage to the target bacteria, grown in the target bacteria, detectable amounts in samples exposures to the bacteriophage (37) Comprising a step (20) of providing sufficient conditions to produce either the bacteriophage or biological material (97) associated with the bacteriophage, the method comprising:
Applying a sample exposed to the bacteriophage to a lateral flow strip (40) or a SILAS surface (220), wherein either the bacteriophage or a biological substance associated with the bacteriophage when present in exposures sample, the change at least a portion of the lateral flow strip or SILAS surface (46,240) of the color, the lateral flow strip, a perpendicular flow of the flow strip A sample pad (43) is included along a second line having one line and a second antibody, the first line comprising a first antibody capable of binding to the bacteriophage or biological material associated with the bacteriophage, and a color marker Having a mixture with colored beads as the first line and Two lines are formed parallel to the, the SILAS surface colors that have been coated with an antibody that changes, process, and as an indication of the presence or absence of the target bacteria, determining the presence or absence of the color change,
A method characterized by. The coating step, the samples exposures to the bacteriophage, characterized in that it comprises the step of applying to the lateral flow strip (40), said lateral flow strip, a third line having a third antibody The method of claim 1 further comprising: the third line is parallel to the first line and the second line . The method of claim 1, wherein the bacteriophage (70) is genetically modified (300, 310, 320, 331). 4. The method of claim 3 , wherein the bacteriophage has been genetically modified (300) to enhance a desired property of an infection process. 4. The method of claim 3 , wherein the bacteriophage has been genetically modified (310) to overexpress a detectable biomarker. 4. The method of claim 3 , wherein the bacteriophage has been genetically modified (320) to express an enzyme. 4. The method of claim 3 , wherein the bacteriophage has been genetically modified (331) to express a target on a capsid protein (72). A method for determining the resistance or sensitivity to antibiotics target bacterial, method, hereinafter:
(B) dividing the sample into a first sample and a second sample;
(C) adding the antibiotic to the second sample;
(D) said first sample and a second for each sample to implement the method of claim 1 step; compare the results of and (e) with the said first sample and said second sample, the Determining the resistance or sensitivity of the target bacteria to antibiotics;
A method characterized by. A system for determining the presence or absence of a target bacterium in a test sample, the system comprising:
  A bacteriophage specific for the bacterium (268);
  A sample pad (43) comprising colored beads (45) bound to a first antibody (44) against said bacteriophage; and
  A porous strip (64) in contact with the sample pad, the porous strip having a detection line (46) comprising a second antibody (47) embedded in the porous strip; Is a porous strip that is perpendicular to the direction of flow in the porous strip;
Including the system. The system of claim 9, further comprising a bacteriophage incubation vessel (260) containing the bacteriophage.

Images