| Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells -> Monitor Keywords |
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Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophageMethod for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070148638, Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60/319,184, entitled "METHOD OF DETECTING LOW CONCENTRATIONS OF A TARGET BACTERIA THAT USES PHAGES TO INFECT TARGET BACTERIAL CELLS" and filed by Angelo J. Madonna and Kent J. Voorhees on Apr. 12, 2002, and U.S. Non-provisional application Ser. No. 10/249,452, entitled "METHOD OF DETECTING LOW CONCENTRATIONS OF A TARGET BACTERIA THAT USES PHAGES TO INFECT TARGET BACTERIAL CELLS" and filed by Angelo J. Madonna and Kent J. Voorhees on Apr. 10, 2003, which applications are incorporated by reference into this application in their entireties. FIELD OF THE INVENTION [0002] The present invention relates to a method for detecting low concentrations of a target bacterium in a liquid mixture that uses bacteriophages to infect target bacterial cells. BACKGROUND OF THE INVENTION [0003] Standard microbiological methods have relied on substrate-based assays to test for the presence of specific organisms (Bordner, et al. 1978). These techniques offer very high levels of selectivity but are hindered by the requirement to first grow or cultivate pure cultures of the targeted organism, which can take 24 hours or longer. This time constraint severely limits the effectiveness to provide a rapid response to the presence of virulent strains of microorganisms. [0004] Molecular biology techniques are quickly gaining acceptance as valuable alternatives to standard microbiological tests. In particular, serological methods have been widely employed to evaluate a host of matrices for targeted microorganisms (Kingsbury & Falkow 1985; Wyatt et al. 1992). These tests focus on using antibodies to first trap and then separate targeted organisms from other constituents in complicated biological mixtures. Once isolated, the captured organism can be concentrated and detected by a variety of different techniques that do not require cultivating the biological analyte. [0005] One very popular approach, termed immunomagnetic separation (IMS), involves immobilizing antibodies to spherical, micro-sized paramagnetic beads and using the antibody-coated beads to trap targeted microorganisms from liquid media. The beads are easily manipulated under the influence of a magnetic field facilitating the retrieval and concentration of targeted organisms. Moreover, the small size and shape of the beads allow them to become evenly dispersed in the sample, accelerating the rate of interaction between bead and target. These favorable characteristics lead to reductions in assay time and help streamline the analytical procedure making it more applicable for higher sample throughput and automation. [0006] Downstream detection methods previously used with IMS include ELISA (Cudjoe, et al. 1995), dot blot assay (Skjerve et al. 1990), electrochemiluminescence (Yu and Bruno 1996), and flow cytometry (Pyle, et al. 1999). Although these tests provide satisfactory results, they are laborious to perform and give binary responses (yes/no) that are highly susceptible to false-positive results due to cross-reactivity with non-target analytes. Recently reported is a rapid method for identifying specific bacteria from complex biological mixtures using IMS coupled to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS)(Madonna et al. 2001). This approach allowed a variety of matrices to be evaluated for the presence of a Salmonella species within a total analysis time of 1 hour. Moreover, the developed procedure required little sample processing, was relatively easy to perform, and the molecular weight information obtained made it possible to discriminate between signals from the target bacteria and signals from cross-reacted constituents. [0007] MALDI-TOF-MS is a proven technique for identifying whole cellular microorganisms (Holland et al (1996); van Barr 2000; Madonna et al. 2000). In principle, MALDI is a `fingerprinting` technique where mass spectra featuring varying distributions of protein signals are generated. The signature profiles that are produced, due to inherent differences in microbial proteomes, make it possible to discriminate between organisms down to the strain level (Arnold and Reilly 1998). The MALDI-TOF technique coupled with IMS includes, in one embodiment, mixing immunomagnetic beads specific to the target bacteria with the liquid mixture that may contain the target bacteria for a short incubation period (e.g., 20 min). Any target bacteria captured by the beads are washed twice, re-suspended in deionized H.sub.2O, and directly applied onto a MALDI sample probe. The target bacteria-bead complex is then overlaid with a micro-volume of matrix solution and dried at room temperature. Irradiation of the resulting crystalline mass with a high intensity laser promotes the liberation and ionization of intact cellular proteins that are subsequently detected by a TOF mass spectrometer. The resulting mass spectrum is then interrogated for definitive mass peaks that signify the presence of the target bacteria. SUMMARY OF THE INVENTION [0008] The invention is directed to a method for determining if a target bacterium is present in a liquid solution when the target bacterium is or may be present in a low concentration that is at or near the detection limit for a particular detection technology. As used herein, the term "target bacterium" refers to a specie of species of bacteria. In turn, the invention is applicable to situations in which it is desirable to determine whether a target bacterium (e.g., E. coli) is present in a liquid solution when the number of target bacterium per unit volume of solution (i.e., the concentration of the target bacterium) is or may be below the detection limit for a particular detection technology. In some instances, a plurality of target bacterium may be referred to as the target bacteria. [0009] In one embodiment, the process comprises using a biological amplification procedure in which bacteriophages for the target bacterium are applied to the liquid solution. (Bacteriophages are viruses that infect bacteria and in the process produce many progeny. Structurally, the bacteriophage consists of a protein shell (capsid) that encapsulates the viral nucleic acid. The capsid is constructed from repeating copies of the same protein. Bacteriophages are able to infect specific bacterial cells and because of the multiplication of the genetic material, the cells eventually burst releasing millions of copies of the original phage.) The bacteriophages and any of the target bacterium present in the liquid solution are allowed to incubate. During the incubation period, the bacteriophages will multiply by infecting target bacterium present in the solution. More specifically, the bacteriophage replicates numerous copies of itself in an infected target bacterium. Eventually, the infected target bacterium lyses and the replicated or progeny bacteriophages are released into the liquid solution. The solution is then analyzed to determine if a biomarker for the bacteriophage is present, thereby indirectly indicating that the target bacterium is present in the liquid solution. Possible analysis techniques comprise mass spectrometry techniques, such as MALDI-MS and electro-spray ionization-MS techniques. [0010] To assure that the detection of a biomarker for the bacteriophage indicates that the target bacterium is present in the liquid solution, a concentration of the bacteriophage is applied to the liquid solution that is below the detection limit for the biomarker for the bacteriophage for whatever analysis technique is employed. This assures that if the biomarker for the bacteriophage is detected by the analysis technique, the detectable concentration of the biomarker is attributable to the replication of the bacteriophage by the target bacterium present in the liquid solution. In certain situations, the use of such a concentration of bacteriophage has a multiplicity of infection ("MOI") (i.e., ratio of infecting bacteriophages to target bacterium) that is too low to rapidly produce a sufficient concentration of bacteriophages or biomarkers for the bacteriophage for detection. [0011] Another embodiment of the process addresses this problem by adding a very high concentration of the bacteriophage to the liquid solution, thereby assuring a high MOI. In this case, the concentration of the bacteriophage added to the solution may exceed the detection limit of whatever analysis technique is employed to detect the bacteriophage or biomarker of the bacteriophage. Consequently, the process applies parent bacteriophage to the solution that can distinguished from any progeny bacteriophage resulting from the infection of target bacterium in the mixture. If the distinguishable progency bacteriophage or a distinguishable biomarker of the progeny bacteriophage are present, this indicates that the target bacterium is present in the solution. [0012] In one embodiment, the parent bacteriophage (i.e., the bacteriophage initially applied to the solution) are "tagged" so that whatever analysis technique is employed is inherently capable of distinguishing the parent bacteriophage or parent bacteriophage biomarkers from the progeny bacteriophage or biomarkers for the progency bacteriophage. For example, if a mass spectral analysis technique is employed, the parent bacteriophage are "tagged" with a substance that alters or shifts the mass spectrum of the parent bacteriophage relative to the progeny bacteriophage, which will not inherit the "tag" from the parent bacteriophage. For example, a biotinylated bacteriophage can be employed as a parent bacteriophage and will have a different mass spectrum than the progeny bacteriophage produced by the biotinylated bacteriophage infecting target bacterium present in the solution. Other "tags" can be employed for other types of analytical techniques. [0013] In another embodiment, the parent bacteriophage possesses a characteristic that allows the parent bacteriophage to be separated from the progeny bacteriophage in the liquid solution prior to analysis, thereby assuring that most, if not all of the bacteriophages present in the liquid solution after separation are progeny bacteriophage resulting from the replication of the parent bacteriophage by target bacteria present in the liquid solution. In one embodiment, the parent bacteriophages initially applied to the liquid solution are biotinylated bacteriophages. Biotinylated bacteriophages are highly attracted to strepavidin. This attraction is exploited to separate the biotinylated bacteriophage from progeny bacteriophage resulting from replication of the biotinylated bacteriophage by target bacterium present in the mixture. [0014] In one embodiment, the biotinylated bacteriophage are attached to a strepavidin coated probe. Consequently, separation of the biotinylated bacteriophage from the liquid solution after the incubation period is accomplished by removing the probe from the liquid solution. In another embodiment, strepavidin-coated magnetic beads are applied to the liquid solution. The beads are used to pick up the biotinylated bacteriophage. The beads are then separated from the liquid solution using a magnet. In yet another, embodiment a strepavidin coated probe (e.g., a slide) is applied to the liquid solution after the incubation period. The biotinylated bacteriophage adhere to the probe and then the probe is separated from the liquid solution. [0015] Yet a further embodiment of the invention recognizes that the liquid solution in which the target bacterium may be present is or may be a complex mixture that includes biological material that makes the detection of the bacteriophage or biomarker for the bacteriophage more difficult or reduces the reliability of the information provided by the detection technology employed. For instance, when a mass spectrometry detection methodology is employed, the complex mixture may produce a signal in which the biomarker associated with the bacteriophage is obscured or, stated differently, has a low signal-to-noise ratio. To address this possibility, the liquid solution is subject to a purification step in which target bacterium that are present in the liquid solution are separated from the remainder of the solution. In one embodiment, immuno-magnetic separation ("IMS") is utilized to separate target bacterium present in the liquid solution from the remainder of the solution. In one particular embodiment, magnetic beads are coated with an antibody for the target bacterium. The antibodies pick up the target bacterium present in the liquid mixture and then a magnet is used to separate the beads from the remainder of the liquid solution. The beads and any adhering target bacterium are then subjected to the biological amplification process and analysis. It should be appreciated this purification step also addresses the possibility that feral versions of the bacteriophage may be present in the liquid solution and that such versions could produce a false positive if the liquid solution was not subjected to a purification step. [0016] If feral versions of the bacteriophage are not of concern, the purification step can be implemented after the biological amplification process. In this embodiment, the purification step involves separating the bacteriophages and the liquid solution, rather than separating the target bacterium and the liquid solution. In one embodiment, an IMS is used in which magnetic beads are coated with an antibody for the bacteriophage. The beads pick up the bacteriophages present in the solution and then a magnet is used to separate the beads from the remainder of the solution. [0017] In another embodiment of the invention, the analysis step comprises using MS/MS analysis to determine if a biomarker for the target bacterium is present. The use of MS/MS analysis produces a highly reliable indication of the presence of a biomarker for a target bacterium. As a consequence, at least in some cases, the use of MS/MS analysis renders the need for a purification step unnecessary. [0018] In yet another embodiment, the invention is directed to a process for detecting low concentrations of a target bacterium in complex mixtures. In one embodiment, the process comprises using an IMS procedure to isolate at least some of a target bacterium that may be present in a liquid mixture. The process further includes employing a biological amplification procedure in which a low titer or concentration of bacteriophages for the target bacterium are applied to at least some of the target bacterium that has been isolated by the IMS procedure. The mixture of bacteriophages and any of the target bacterium that has been isolated is allowed to incubate. If at least a certain concentration of the target bacterium is present, the bacteriophages will multiply during the incubation period such that a high titer or concentration of bacteriophages will be present in the mixture and detectable by MALDI-TOF-MS analysis. If no or only a small number of the target bacterium is present, there will be a low concentration of bacteriophages present in the mixture that will not be reasonably detectable by MALDI-TOF-MS analysis. Following incubation, a MALDI-TOF-MS analysis is performed on the incubated mixture of bacteriophages and target bacterium. The resulting mass spectrum is analyzed to determine if a protein that is associated with the bacteriophages is present. If the protein for the bacteriophage is detected, then it can be concluded that at least a low concentration of the target bacterium is present in the mixture. [0019] It should also be appreciated that the method of the invention is capable of detecting low concentration of a target bacterium regardless of the manner in which the bacterium was grown or propagated. BRIEF DESCRIPTION OF THE DRAWINGS Continue reading about Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells... 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