EP1789572A2 - Detection de micro-organismes au moyen d'une amplification bacteriophage - Google Patents

Detection de micro-organismes au moyen d'une amplification bacteriophage

Info

Publication number
EP1789572A2
EP1789572A2 EP05856796A EP05856796A EP1789572A2 EP 1789572 A2 EP1789572 A2 EP 1789572A2 EP 05856796 A EP05856796 A EP 05856796A EP 05856796 A EP05856796 A EP 05856796A EP 1789572 A2 EP1789572 A2 EP 1789572A2
Authority
EP
European Patent Office
Prior art keywords
bacteriophage
sample
microorganism
nucleic acid
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05856796A
Other languages
German (de)
English (en)
Inventor
John H. Wheeler
Jon Rees
G. Scott Gaisford
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MicroPhage Inc
Original Assignee
MicroPhage Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MicroPhage Inc filed Critical MicroPhage Inc
Publication of EP1789572A2 publication Critical patent/EP1789572A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Definitions

  • the invention solves the above problems, as well as other problems of the prior art, by providing methods and apparatus for detecting living microorganisms using the principle of phage amplification combined with the use of a bacteriophage progeny biological substance selected from bacteriophage nucleic acid (DNA/RNA), bacteriophage protein, and bacteriophage intermediate protein, as an indicator of the presence of the microorganism.
  • bacteriophage intermediate protein is protein that is created in the bacteriophage replication process that does not become part of the replicated bacteriophage.
  • Bacteriophage nucleic acid can include nucleic acid injected by the parent bacteriophage or nucleic acid formed in the replication process.
  • the invention also solves the above problems, as well as other problems, by mobilizing the microphage, either by coupling the bacteriophage to mobile substrates, or by not coupling the phage to a substrate at all, both of which permit more thorough contact of a sample, particularly insitu samples, to the parent bacteriophage, thereby increasing the signal derived from samples with small numbers of the target microorganisms.
  • the measuring of a reference level comprises taking a reference sub-sample from the bacteriophage exposed sample and performing a reference assay on the reference sub-sample before any bacteriophage amplification has occurred in the reference sub-sample, and wherein the test level is measured for the bacteriophage exposed sample after some of the bacteriophage replication process has occurred.
  • the combining comprises tagging the parent bacteriophage.
  • the assaying comprises isolating the tagged parent bacteriophage from the bacteriophage exposed sample.
  • the tagging comprises attaching the parent bacteriophage to a mobile substrate that can be added to or retrieved from the sample.
  • the isolating comprises removing the tagged, parent bacteriophage from the sample.
  • FIG. 6 illustrates a second embodiment of the invention wherein phage are added to the sample to give an initial concentration below the detection limit and where the phage are dissociated such that phage subcomponent biomarkers are detected;
  • FIG. 11 illustrates an exemplary embodiment of an assay process according to the invention utilizing immunomagnetic separation and bacteriophage nucleic acid detection.
  • bacteriophage specific to a species of bacteria is added to a suspension suspected of containing the targeted bacterium. If this bacterium is present, the bacteriophage infects the cell and replicates numerous copies of itself. Lysing of the bacterium, either naturally or through the use of a lysing agent, releases the replicated phages into the surrounding medium.
  • DNA/RNA bacteriophage nucleic acid
  • the use of a bacteriophage nucleic acid (DNA/RNA) as a marker permits gei electrophoresis, oligo capture, fluorescence labeling, and other nucleic acid detection systems to be used to detect the bacteria.
  • the DNA evolves to early mRNAS 155 and early proteins 156, some of which become membrane components along line 157 and others of which utilize bacteria nucleases from host chromosomes 159 to become DNA precursors along line 164. Others migrate along the direction 170 to become head precursors that incorporate the DNA along line 166.
  • the membrane components evolve along the path 160 to form the sheath, end plate, and pins.
  • Other proteins evolve along path 172 to form the tail fibers. When formed, the head releases from the membrane 151 and joins the tail sheath along path 174, and then the tail sheath and head join the tail fibers at 176 to form the bacteriophage 70.
  • the early proteins 156, precursor proteins along paths 172 and 174, and any other protein that is formed during the process of replication but is not present in the parent or final replicated bacteriophage is referred to herein as intermediate proteins.
  • the phage nucleic acid, the intermediate proteins, and the phage proteins associated with the progeny phage are particularly useful as indicators of the presence of the target microorganism.
  • Some bacteriophage, called lytic bacteriophage rupture the host bacterium, shown at 180, releasing the progeny phage into the environment to seek out other bacteria. Lytic phages are typically used in the method disclosed herein.
  • the INCUBATE process 20 is shown in FIG. 2.
  • the parent phage 18 infects 32 the target bacteria 14 by attaching themselves to cell walls of the target bacteria and injecting the viral nucleic acid to create infected bacteria 23.
  • Replication 34 of progeny phage as indicated in FIG. 10 then proceeds within the host bacteria. In some embodiments, the replication of progeny page is permitted to proceed to completion. If lytic phages are used, the host ruptures in a lysis process 36 releasing the progeny phage 37 into the test sample where they may infect other target bacteria. This incubation process may proceed for one or more cycles of infection, amplification, and lysis.
  • a bacterial lysome 22 for the particular microorganism is added in process 21 , preferably prior to complete formation of the progeny phage, which, in process 25, causes the cell walls of essentially all the particular microorganism, such as a bacterium, present in the test sample 24 to rupture, thereby releasing essentially all bacteriophage progeny biological substances 97, including nucleic acid injected into the bacterium by the parent bacteriophage, bacteriophage intermediate proteins, progeny bacteriophage nucleic acid, and progeny bacteriophage phage proteins, and also all microorganism internal biological materials, including microorganism DNA, contained therein.
  • bacteriophage progeny biological substances includes nucleic acid 154 injected into the microorganism by the parent phage, but does not include complete progeny bacteriophage.
  • the parent phage added to the raw sample and the progeny phage, if produced during the incubation process, are identical. This means that, even if there are no target bacteria in the test sample, there will still be phage present during detection process 28 that might give rise to an associated background signal. Generally, since phage are much more resistant to lysing agents than bacteria, phage nucleic acid should not be present. However, it may be possible that one or more phage may lyse due to natural defects in phage. A method of solving this problem is to control the initial concentration of parent phage in the test sample such that the background signal due to any phage nucleic acid produced is undetectable in detection process 28.
  • Another way to reduce the background signal is to detect one or more intermediate proteins in the process 28.
  • these intermediate proteins are distinguishable from the proteins of the parent phage.
  • the parent phage do not provide a background signal for these intermediate proteins.
  • Any detection method or apparatus that detects phage nucleic acid or phage protein will suffice for this method 28.
  • Preferred methods for detection of phage nucleic acid are gel electrophoresis, oligo capture, fluorescence labeling, colorimetric methods, and other nucleic acid detection systems.
  • Preferred methods for detection of phage proteins include immunoassay methods utilizing antibody-binding events to produce detectable signals, ELISA, flow cytometry, western blots, aptamer-based assays, radioimmunoassay, immunofluoresence, lateral flow immunochromatography (LFI), matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF-MS), referred to herein as MALDI, the use of a SILAS surface which changes color as a detection indicator, and other protein detection methods.
  • MALDI matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry
  • FIG. 3 is a flow chart illustrating the preferred processes 300 of the invention, including preferred optional processes, in more detail.
  • the process 300 begins with the preparation of a sample at 302. Usually this will involve the providing of a raw sample to be tested, which sample will usually include complex insitu matrixes. Generally, a portion of the raw sample will be diluted with a liquid such as sterile water to provide a suitable volume of prepared insitu sample. This sample is then combined at 304 with a suitable quantity of bacteriophage.
  • a feature of the invention is that a very large quantity of bacteriophage, such as 10 6 to 10 10 bacteriophage, may be added while still providing an accurate result.
  • 10 7 to 10 9 bacteriophage may be added.
  • from 5 X 10 7 to 5 X 10 8 bacteriophage are added.
  • This large quantity of bacteriophage greatly accelerates the process.
  • these large quantity of bacteriophage may be added in processes in which the reference process 330 is used, or processes in which the parent phage is isolated from the progeny phage, or in processes in which the microorganism but not the phage is dissociated.
  • the phage may be added by combining a solution of phage with the sample solution, combining a phage containing substrate with the sample, or any other convenient method of adding the phage to the sample or adding the sample to the phage.
  • the process then preferably goes directly to subprocess 328 via path 306, which subprocess 328 will be discussed below, or, optionally, it goes to an isolation, purification, and/or concentration process 308.
  • the isolation, purification, and/or concentration process 308 begins with process 310 in which the bacteria is captured.
  • Process 310 preferably comprises capturing the microorganism with either the phage which were added in process 304, or with a non-phage capturing medium, such as antibodies.
  • the phage that are combined in process 304 are preferably attached to a substrate, which may be a plate or other large surface or structure, or may be a probe made of mesh, magnetic beads, or other mobile structure that can be stirred in the sample prepared in step 302.
  • An optional process is illustrated by dashed lines 325 and 326.
  • the isolation, purification, and/or concentration process 308 takes place prior to the combining process 304.
  • the capture process 310 takes place after the sample preparation process 302 as shown by path 325.
  • the Place In Sub-Sample Process 324 the combining process 304 is performed, as indicated by path 326. This optional process then proceeds directly to the incubate process 328 via path 306.
  • the subprocess 360 may be selected from immunoassay methods utilizing antibody-binding events, ELISA, flow cytometry, western blots, aptamer-based assays, radioimmunoassay, immunofluoresence, and lateral flow immunochromatography (LFI).
  • Other protein detection methods are matrix- assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI- TOF-MS), referred to herein as MALDI, and the use of a SILAS surface which changes color as a detection indicator, or any other protein detection process.
  • MALDI- TOF-MS matrix- assisted laser desorption/ionization with time-of-flight mass spectrometry
  • SILAS surface which changes color as a detection indicator, or any other protein detection process.
  • a reference process 330 is initiated as shown by path 305.
  • a reference sample is separated from the test sample in process 331. If the optional isolation, purification, and/or concentration process 308 is performed, and it is also desired to include the reference process 330, then the reference sample is separated after process 324, as shown by path 327.
  • Process 331 generally comprises transferring a suitable portion of the test sample, preferably half of it, to a separate container or other medium. The process 300 may then proceed directly to process 338 via path 332, or, optionally, proceed to process 334.
  • test sample 50 One or more drops of a test sample 50 are added to the sample pad.
  • the test sample flows along the lateral flow strip 40 toward the absorbent pad 52 at the opposite end of the strip.
  • phage protein particles flow along the conjugate pad toward the membrane, they pick up one or more of the first antibody-bead conjugates forming protein-bead complexes.
  • protein-bead complexes move over row 46 of second antibodies, they form an immobilized and concentrated first antibody-bead-protein-second antibody complex. If enough protein-bead complexes bind to the row 46 of immobilized second antibodies, a colored line becomes visible to the naked eye. A visible line indicates that the target bacteria were present in the raw sample.
  • FIG. 6 illustrates a second embodiment 90 of a method to detect target bacteria according to the invention, which method 90 has enhanced sensitivity.
  • Processes 12, 20, and optional process 21 consisting of ADD PHAGE, INCUBATE, and LYSE BACTERIA are identical to the corresponding processes described in association with FIGS. 1 and 2, though it should be understood that the full incubation process of FIG. 2 is preferred here.
  • the test sample contains an abundance of phage nucleic acid and phage protein particles if target bacteria were present in the raw sample.
  • process 94 of the second embodiment 90 As shown in FIG. 6, process 94 of the second embodiment 90,
  • This potential limitation can be overcome if the signal associated with the parent phage can be eliminated or significantly reduced such that higher concentrations of parent phage can be utilized - MOIs greater than 5. It can also be overcome if the signal due to the progeny phage is enhanced, such as by the use of genetically enhanced phage, both of which are discussed in detail herein.
  • the tagged parent phage are segregated from the progeny bacteriophage by extracting or substantially removing them from the test sample or otherwise isolating the parent phage from the progeny phage such that they cannot interfere with or contribute to the analyzed signal. If the tagged parent phages are attached to a physical substrate when added to the raw sample in process 105, then the substrate and associated parent phage are preferably physically removed from the test sample in process 114. Biotinylated phage that are not attached to a physical substrate also can be readily segregated or removed from the test sample.
  • NUCLEIC ACID is to analyze the test sample to detect nucleic acid injected by the parent phage and nucleic acid associated with the forming progeny phage as a surrogate marker for target bacteria present in the raw sample.
  • the detection means used with this embodiment are identical to those described with respect to processes 28 and 29 of the embodiments 10 and 90, respectively, as illustrated in FIGS. 1 and 6, respectively. As with the earlier embodiments, any suitable detection method or apparatus may be used.
  • FIG. 8 illustrates a fourth embodiment 120 of a method to detect target bacteria in a sample, in which method 120 the sensitivity is enhanced.
  • Embodiment 120 is a combination of the methods taught in embodiments 90 and 100.
  • Processes 105, 107, 108, and 114 are identical to those taught with embodiment 100 and illustrated in FIG. 7, i.e., ADD PHAGE, INCUBATE, optionally LYSE BACTERIA, and EXTRACT TAGGED PHAGE, respectively.
  • the phage replication is preferably permitted to go to completion in the INCUBATE step.
  • embodiment 120 incorporates tagged parent phage in process 105 and a parent phage removal or segregation process in process 114.
  • a phage dissociation agent 122 is added to the test sample 124 as taught in process 94 of embodiment 90 and illustrated in FIG. 6.
  • the tagged parent phage is physically removed from the test sample in process 114 rather than simply segregated so that it will not be exposed to the phage dissociation agent in process 121.
  • the test sample 124 contains only progeny phage, and the dissociated test sample 126 will contain biological marker material, including phage nucleic acid, only from progeny phage.
  • DETECT PHAGE NUCLEIC ACID process 130 of the embodiment 120 illustrated in FIG. 6 is preferably the same as any of the processes 28, 99, and 116 of the earlier embodiments.
  • FIG. 9 illustrates a method 140 by which any of the embodiments of the invention can be used to detect a target bacterium, and if present, determine if it is resistant to one or more antibiotics.
  • a sample 142 that may contain the target bacterium is divided into two: a first Sample A, indicated by 144, and a second Sample B, indicated by 145.
  • a first antibiotic 146 is added to Sample B whereupon the target bacteria in Sample B are killed if they are not resistant to the first antibiotic.
  • Samples A and B are then analyzed at 148 and 149 to detect the presence of viable target bacteria in each, giving Result A and Result B. Any of the methods taught in this invention can be used for these analyses.
  • the phage and target are incubated for a short time, and the target is then dissociated or lysed to release phage nucleic acid, protein, or other bacteriophage progeny biological substance.
  • the lysing is active lysing but may also be natural lysing.
  • a drop of the water with bacteriophage progeny biological substance is then transferred in process 472 to suitable test medium 466 which may be a solid, a fluid, or a device, such as a flow strip, which can be utilized for the desired detection process .
  • suitable test medium 466 may be a solid, a fluid, or a device, such as a flow strip, which can be utilized for the desired detection process .
  • the biological substance is nucleic acid, it may be amplified at 474 using PCR, SDA, Rolling Circle, etc.
  • the bacteriophage progeny biological substance is then detected in process 478.
  • the beads 450 may be coated with a target-specific bacteriophage.
  • the incubation is kept very short, for example 1 to 10 minutes, while the beads are stirred vigorously in the bacterial mixture.
  • additional bacteriophage in the solvent in process 464 is not necessary.
  • the entire process from the addition of the beads in process 441 to the dissociation of lysing of the target in process 464 should be short, preferably no more than 20 minutes.
  • this time can be longer also.
  • the protocol described herein is intended to reduce, if not eliminate, the need for nucleic acid amplification techniques such as PCR, SDA, Rolling Circle, etc.
  • concentration of bacteriophage nucleic acid after amplification according to the invention is often great enough to be detected under most conventional detection systems without additional amplification using PCR, SDA, Rolling Circle, etc. In such cases, the protocol will reduce costs significantly, and be less affected by temperature, cross contamination, probe sequence errors, and complex extraction techniques.
  • the nucleic acid or protein being detected is that of the infecting bacteriophage and represents an amplification factor of up to 20,000 copies per infected pathogen.
  • PCR methods potentially amplify non-specific nucleic acids in a sample
  • an additional 10 amplification cycles result in 1000 times more non-specific nucleic acid signal than does the two-step phage- amplification/PCR protocol.
  • the contaminating nucleic acids result in significantly higher rates of false positives.
  • the signal-to-noise ratio is many times higher with the two-step phage-amplification/PCR protocol than with the one-step PCR protocol. The higher signal-to-noise ratio has the added benefit of lower detection limits for the bacterium of interest.
  • the two-step amplification protocol consisting of a phage amplification step followed by PCR amplification of the phage nucleic acids is more sensitive, selective, and can distinguish between viable and non-viable bacteria.
  • the samples that potentially contain the target microorganism(s) are complex organic mixtures containing large numbers of proteins, enzymes, lipids, nucleic acids, etc.
  • One such application is detecting bacterial contamination in foods such as raw beef or chicken where the sample may contain ground up meat. Detecting specific bacterial nucleic acids in such organically complex samples is challenging. It is highly advantageous to isolate the nucleic acids from such a matrix prior to amplification and detection.
  • the phage nucleic acid amplification method can be used to isolate the desired nucleic acids from a complex organic matrix that may contain target microorganism(s). An example of such a process follows.
  • the tagged, parent bacteriophage then is added to the sample and allowed to incubate sufficiently long enough to allow some or all of the target microorganisms in the sample to bind to tagged, parent bacteriophage but not long enough for the bacteriophage infection cycle to be completed, resulting in lyses of the target microorganisms.
  • the tagged, parent bacteriophage would be added to the sample for a period of time less than 90 minutes.
  • This second process may be considered a capture process where the tagged, parent bacteriophage are used to capture some or all of the target microorganisms in the sample.
  • the nucleic acid in the reagent solution may be amplified using conventional amplification methods such as PCR. If the optional cleaning process above has been performed, the reagent solution will essentially contain nucleic acids from parent and progeny bacteriophage and from the lysed target microorganisms captured in process 2 above. If optional process 6 above is also performed, then the only nucleic acids present in the reagent solution will be that of progeny bacteriophage and of captured, target microorganisms.
  • the reagent sample is then assayed to determine the presence or absence of bacteriophage associated nucleic acid as an indication of the presence or absence of the target microorganism in the original sample.
  • bacteriophage are used to capture and isolate the target microorganisms from the complex sample matrix, thereby eliminating many problems associated with PCR assays of raw samples.
  • a background reference assay may be performed by taking a reference sub-sample from the bacteriophage exposed sample and assaying that reference sub-sample before any bacteriophage amplification has occurred in the reference sub-sample.
  • the reference result then can be obtained from this optional assay.
  • the reference assay result is a direct measure of the background signal associated with the bacteriophage exposed sample. Any detectable increase in the signal for the assay performed on the bacteriophage exposed sample as compared to the reference assay result is a direct indication of the presence of the target microorganism in the original sample.
  • United States Patent Application Serial No. 10/823,294 which has been incorporated herein by reference, discloses many aspects of the bacteriophage amplification and detection process, which can be combined with the processes disclosed herein.
  • the detection method used is the detection of a bacteriophage biological substance, preferably nucleic acid (DNA/RNA), as a marker.
  • DNA/RNA nucleic acid
  • the process of the invention is relatively sensitive, fast, simple, and/or economical as compared to the prior art.

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Abstract

L'invention concerne une méthode de détection de la présence ou de l'absence d'un micro-organisme cible dans un échantillon à tester. Ladite méthode consiste à mélanger l'échantillon avec un bactériophage capable d'infecter le micro-organisme cible afin de créer un échantillon exposé à un bactériophage, à fournir certaines conditions à l'échantillon qui permettent au bactériophage d'infecter le micro-organisme cible de manière à engendrer un acide nucléique de bactériophage injecté, un acide nucléique de bactériophage supplémentaire, une protéine de bactériophage intermédiaire ou une protéine de bactériophage supplémentaire, et à doser l'acide nucléique ou la protéine en vue de déterminer la présence ou l'absence du micro-organisme cible. Le micro-organisme est, de préférence, dissocié au lysé avant la réalisation du processus de réplication du bactériophage. Cet échantillon est un échantillon in situ et le micro-organisme est isolé de la matrice in situ. Le dosage consiste à comparer l'acide nucléique de test ou le niveau de protéine à un niveau de référence.
EP05856796A 2004-06-07 2005-06-07 Detection de micro-organismes au moyen d'une amplification bacteriophage Withdrawn EP1789572A2 (fr)

Applications Claiming Priority (3)

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US57784504P 2004-06-07 2004-06-07
US60794104P 2004-09-07 2004-09-07
PCT/US2005/020248 WO2006083292A2 (fr) 2004-06-07 2005-06-07 Detection de micro-organismes au moyen d'une amplification bacteriophage

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US8216780B2 (en) 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
JP2010508044A (ja) * 2006-10-31 2010-03-18 マイクロファージ・インコーポレーテッド 潜在的に交差反応性の生物の選択阻害による、強化されたバクテリオファージベース診断アッセイの方法及び装置
AU2008265989B8 (en) 2007-06-15 2012-01-12 Microphage Incorporated Method of detection of microorganisms with enhanced bacteriophage amplification
US9441204B2 (en) 2008-04-03 2016-09-13 Colorado School Of Mines Compositions and methods for detecting Yersinia pestis bacteria
WO2012158041A1 (fr) 2011-05-18 2012-11-22 Rna Holding B.V. Procédés et kits diagnostiques pour déterminer la présence d'un microorganisme dans un échantillon
WO2023122608A1 (fr) * 2021-12-21 2023-06-29 Adaptive Phage Therapeutics, Inc. Dosage pour mettre en correspondance un phage avec des bactéries

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US5888725A (en) * 1992-09-22 1999-03-30 The Secretary Of State For The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Method for identifying target bacteria
EP1540018B1 (fr) * 2002-04-12 2010-05-26 Colorado School Of Mines Procede de detection de faibles concentrations d'une bacterie cible qui utilise des phages pour infecter des cellules bacteriennes cibles
US20050003346A1 (en) * 2002-04-12 2005-01-06 Colorado School Of Mines Apparatus and method for detecting microscopic living organisms using bacteriophage

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