WO1993017129A1 - Methode et necessaire a diagnostic pour la determination de bacteries - Google Patents

Methode et necessaire a diagnostic pour la determination de bacteries Download PDF

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Publication number
WO1993017129A1
WO1993017129A1 PCT/US1993/001627 US9301627W WO9317129A1 WO 1993017129 A1 WO1993017129 A1 WO 1993017129A1 US 9301627 W US9301627 W US 9301627W WO 9317129 A1 WO9317129 A1 WO 9317129A1
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Prior art keywords
bacteriophage
bacteria
specific
particles
specific bacteria
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PCT/US1993/001627
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English (en)
Inventor
Paul W. Judkins
E. Birch Ruth
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Judkins Paul W
Birch Ruth E
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Publication of WO1993017129A1 publication Critical patent/WO1993017129A1/fr

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    • 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
    • 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
    • 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

Definitions

  • This invention relates to microbiology, and more particularly, to a method and kit using bacteriophage for the determination of the presence of organisms of a specific bacteria type in a sample.
  • Foodbome disease has been estimated to cause at least six million illnesses and more than a billion dollars in economic losses each year in the United States. There are an estimated 800,000 to 4,000,000 Salmonella infections in the United States each year, and at least 500 deaths. At least two million cases of Campy lobacter infections are estimated to occur each year in the United States, with estimates of deaths associated with Campylobacter infections ranging from 200-800 annually. The increase in the number of Salmonella outbreaks has been unrelenting. From the period 1973- 1975 to 1985-1987, there was a 75% increase in the proportion of bacterial outbreaks caused by Salmonella, and outbreaks of Clostridium botulinum intoxication, Listeria infections, and Salmonella infections accounting for 75% of the known deaths. Testimony of the Director, Div. of Bacterial and Micrological Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, Dept. of Health and Human Services, before the Committee of Human and Labor Resources of the United States Senate on June 28, 1991.
  • Campylobacter Of hospitalized cases of Salmonellosis, up to 25% may be invasive. entering the blood stream and infecting other organ systems. The very young, the elderly, and immunocomprised people are especially suspectable to such septicemias, which sometimes have fatal consequences. Both Salmonella and Campylobacter can
  • SUBSTITUTE SHEET also lead to a form of arthritis known as reactive arthritis, in about 1-3% of the cases, and can debilitate a person for six months or more.
  • Listeriosis is a psychrotroph which grows well at 2-4°C, where competition by other organisms for nutrients is suppressed.
  • the FDA has estimated that there are 18,050 cases of Listeriosis. with 425 deaths in the United States annually. The most common form of Listeriosis is meningitis. The mortality rate may reach 30% in the newborn, elderly, and people with defective immune systems. Pregnant women with meningitis often abort, and the infant, if living, usually develops a fatal septicemia. The clinical course develops and progresses suddenly, and the fatality rate may .be as high as 70% .
  • Listeria isolation, identification and enumeration methods have improved over the years since its discovery in the 1920s, these methods are still labor intensive and time consuming. The shortest methods take two weeks or more for confirmation of identity.
  • Hemorrhagic Escherichia coli is an organism of particular significance because there is an allowable level of the harmless types of E. coli in certain types of foods.
  • the Center for Disease Control identified hemorrhagic E. coli as the pathogen of an outbreak from hamburgers consumed in a fastfood restaurant chain, and since 1982, this E. coli strain has caused disease outbreaks in nursing homes, schools and other institutions. A test that detects all E. coli and cannot differentiate the hemorrhagic strain is almost useless in preventing foodbome infection by this organism.
  • SUBSTITUTE SHEET widening varieties of food products. These products often have sensitive shelf life, particularly in the case of ready-to-eat meats and dairy products. Rapid and efficient methods of distribution have come into place to deliver, particularly shelf life sensitive products, to points of sale as quickly as possible to preserve most of the product's shelf life. This rapidity with which vast quantities of food products are produced and distributed to the consuming public has outpaced the technology for testing specimens of the food product for bacterial contamination by pathogens. During the three-to-four days needed for a test screening of a food product for bacterial contamination, the food usually has already left the plant and moved through the food distribution system to points of sale, particularly when the product is shelf-life sensitive.
  • test results for a pathogen By the time test results for a pathogen are available, the food may already have been consumed. If test results are positive and necessitate a recall, the recall of the product must then also be directed to the consumer. This may be too late for the prevention of outbreak of a bacterial infection brought on by the pathogen. Even if the product is warehoused pending receipt of microbiological testing results before release for distribution, costs of warehousing for the lengths of time to get screening test results is a substantial cost of food processing which pressures profit margins and increases costs to consumers.
  • HACCP Hazard Analysis and Critical Control Point
  • SUBSTITUTE SHEET microbiological testing of food One is a DNA probe technology. This test requires several complex steps to produce a result. First, selectively enriched broth cultures pass through filters selectively trapping bacteria on their surfaces. Second, a chemical solution dissolves the outer layer of trapped bacteria , releasing cellular DNA into separate uncoiled strands. Third, the DNA strands are chemically fixed to the filter and exposed to a radioactive probe DNA. If the probe locates a complimentary section on the bacterial DNA, it will link and form a hybrid molecule. This hybrid now carrying a radioactive signal, can be measured by scintillation counter that can detect the beta emissions from the radioactive probe DNA.
  • the DNA probe technology is also now offered in a colorimetric assay format where the hybrid forms a color instead of carrying a radioactive signal.
  • the time required for a negative result is three days.
  • the test is sensitive to detect a minimum bacterial organism population typically of one million organisms, or more.
  • EIA enzyme immunoassay
  • the target bacterial antigens If the target bacterial antigens are present, they will bind to the antibodies on the surface of the beads. Washing the beads removes the unbound materials. The beads are then placed in a solution containing peroxidase-conjugated anti-bacterial antibodies. The conjugate binds to the bacterial antigens captured by the antibodies on the surface of the beads. A second washing step removes the unbound conjugate. The beads are then placed in a substrate solution. Appearance of color in the solution suggests the presence of target bacteria in the sample. The time to a negative result is three days. The test is sensitive to discern a minimal bacterial population of 100,000 organisms or greater.
  • SUBSTITUTE SHEET bacterium which is a host for the bacteriophage, was inoculated with the inactivated bacteriophage, a culture of such bacterium was grown, and the amount of plaque formation in the culture was determined, with the degree of plaque formation being inversely proportional to the concentration of the antigen mixed with the antibody conjugate.
  • U.S. Patent 4,797,363 to Teodorescu, et al. described the use of bacteriophage as a carrier for antibodies. According to Teodorescu. the antibodies could be affixed to the bacteriophage by biot ⁇ n-avidin recognition, coating the bacteriophage with avidin or biotin and binding to the antibody that has biotin or avidin, respectively. The bacteriophage could also be tagged with a labeling agent to assist in visualizing reaction of antigen to the antibodies on the bacteriophage.
  • Our invention also makes use of very small solids such as already commercially available latex particles. Such particles previously have been suggested for immunological testing. David, et al. in U.S. Patent 4,486,530, in describing a "sandwich" enzyme immunometric assay technique for determination of the presence of antigenic substances in fluids using monoclonal antibodies, propose that antibodies could be bound to particles of latex.
  • our invention is not limited to testing food, feed or cosmetic stuffs, but has application to
  • the previously described EIA technology the next quickest, commercially available test has a minimum detection sensitivity of roughly 100,000 organisms and takes three days.
  • Our invention provides a method and kit for the determination of the presence of organisms of a specific bacteria type in a sample.
  • Our method includes contacting a sample to be tested for presence of such organisms with both (i) a bacteriophage capable of attaching to such organisms, the bacteriophage having affixed to them one of two members of a specific binding pair, and (ii) a water-insoluble, water- suspendible solid phase carrier particle having affixed to it the second member of the specific binding pair. If organisms of the specific bacteria type are present in the fluid sample, a water-insoluble complex of at least (1) the organism, (2) the bacteriophages, and (3) the carrier particles is formed. The bacteriophage attach to the bacteria for which the
  • SUBSTITUTE SHEET phage are specific, and the carrier particle(s) bind to the phage.
  • This complex if present, is then separated from any particles and bacteriophage in the test mixture not complexed to the specific organism. Testing to detect the complex, if present, is then conducted, if the testing detects the complex, the presence of the specific organisms in the sample is indicated.
  • the complexing or clustering phenomenon just mentioned is an important part of our invention, because it is the foundation for the specificity, sensitivity, and speed of our test. In effect, it identifies and enlarges the presence of a single organism to allow it to be isolated and identified.
  • Either the bacteriophage or the carrier particle has a labeling agent affixed to it.
  • the labeling agent is affixed to the carrier particle.
  • Testing for the complex involves testing ' for the labeling agent.
  • the labeling agent may be affixed to the bacteriophage.
  • the labeling agent is a large polymer, such as a typical enzyme, and particularly where large numbers of such polymers are affixed to the phage, the phage has poor shelf stability, apparently because a dense concentration of large polymers compromises the integrity of the phage head.
  • label detection is visual and requires no instrumentation.
  • the labeling agent is affixed to the carrier particle.
  • a labeled carrier particle in our invention is very shelf stable.
  • a singularly important reason in our invention for affixation of the labeling agent to the carrier particle is because many more of the labeling agents can be affixed to the carrier particle than to the typically much smaller bacteriophage. This facility for loading large numbers of labeling agents to a single carrier particle greatly increases the "signal" strength detectable compared to the signal strength which can be realized by the much smaller number of labeling agents the much smaller bacteriophage can carry.
  • an organism having about a one micron length in its largest dimension a complex larger than three to five microns is formed using carrier particles of the same nominal size as the organism. This permits the organism-bacteriophage-carrier particle complex to be retained on a filtration medium which will pass bacterial organisms other than those for which the phage is specific, as well as unattached phage, carrier particles and other paniculate matter which are not complexed to phage attached to organisms.
  • a container for combining the phage and carrier particles with the sample a suitable filtration medium, preferably a buffered wash solution to wash the filtration medium clean of passable size particles, and a reagent containing a substrate solution which will develop the color in cooperation with the labeling agent preferably on the carrier panicle.
  • HACCP Critical Control Point
  • the complex formed by our invention depends upon attachment of a bacteriophage to the specific bacterial organism to be detected if present, and upon binding of the carrier particle to the attached bacteriophage.
  • the specific binding pair suitably may be a biotin, and avidin pair, or may be an antigen and an antibody to that antigen or similar binding compound pairs.
  • the binding pair is biotin and avidin. and preferably the biotin is affixed to the bacteriophage, rather than to the
  • the concentration of avidinylated carrier particles contacted with a sample being tested for a specific bacteria-type and containing the biotinylated bacteriophage is important. If the concentration of the avidinylated carrier particle is too great, the particles will tend to agglutinate with each other in the reagent and compromise the reagent. Even if less than s ⁇ fficient to agglutinate in the avidinylated carrier particle reagent, the concentration still may be so excessive, upon combination with a solution containing biotinylated bacteriophage, as to result in inter-binding of multiple carrier particles and bacteriophage not attached to target bacteria.
  • the carrier particles are avidinylated and the bacteriophage are biotinylated
  • the carrier particles are provided in suspension in an aqueous medium in a concentration sufficient in the medium to permit formation of the formed complex (of the organism, the bacteriophage and the carrier particle) with the biotinylated bacteriophage, yet insufficient in concentration for agglutination of the particles in the suspension to an extent which prevents their separation from the formed complex (of the organism, the bacteriophage and the carrier particles).
  • the solid carrier particle has a size approximating the size of the organism to be detected; however, the particle size in its largest dimension may vary from as little as 0.2 to as much as 10 times the size of the organism, depending upon the method of separation employed.
  • the carrier particle are about one micron in diameter and are formed of polystyrene latex
  • the carrier particles suitably are in aqueous suspension at a concentration ranging from about 1 x 10 ⁇ to about 1 x 10 ⁇ particles per mL.
  • the biotinylated bacteriophage are in suspension in an aqueous medium, in a concentration of bacteriophage sufficient for a ratio of bacteriophage to organisms of the selected bacteria type effective to produce sufficient of the complex (of the organism, bacteriophage and particle) for detection.
  • the ratio is sufficient for the detection of bacteria present in a sample at a concentration in the range from as little as ten colony forming units (CFU) of organisms and up.
  • the combining bacteriophage is added to the fluid sample being tested for presence of the organisms of the selected bacteria type, whereby if the organism is present, the combining bacteriophage attaches to the organism, and (f) the labeled combining particle is added to the sample, either with or after the combining bacteriophage of step (e), whereby the labeled combining particle binds with the combining bacteriophage, resulting, if the organism of the specific bacteria is present, in a complex of at least the organism, the bacteriophage, and the particle, such complex being of a larger size than the labeled combining particle or the labeled combining particle combined with the bacteriophage unattached to the organism.
  • the method of this invention is particularly adapted to screening for bacteria types selected from the group consisting of Salmonella-type, Listeria-type, Campy lobacter-type, Bacillus-x pe, hemorrhagic Escherichia-type, and Sh ⁇ gella-type bacteria or any other microorganism that is susceptible to phage binding.
  • the method thus involves, in a particular, a determination for the presence in a fluid of organisms of a bacteria type selected from the group consisting of Salmonella- type, Listeria-vypt, Campy lobacter- ype, Bacillus-type, hemorrhagic Escherichia-type, and Shigella-type bacteria, or any other microorganism that is susceptible to phage binding which comprises first contacting a sample of the fluid with (a) a measured amount of an aqueous suspension of biotinylated bacteriophage capable of attaching to organisms of a selected bacteria type, and (b) a measured amount of an aqueous suspension of a water-insoluble, water-suspendible avidinylated solid phase carrier particle labeled with an enzymatic color imetric indicator, the carrier particle having a size approximating one micron ( ⁇ m) in its largest dimension, whereby, if organisms of the selected bacteria type are present in the fluid sample in the predetermined minimum
  • a test mixture is formed containing a complex of at least (1) the organism, (2) the bacteriophage. and (3) the carrier particles, such complex having a size of at least three to five microns in its largest dimension.
  • the test mixture is then deposited onto a top surface of a filtration medium having a pore size approximating, but not larger than, the size of the complex, for example, a pore size of about three to less than five microns for a complex not smaller than five microns.
  • the filtration medium is then washed with a buffered aqueous medium, whereby paniculate matters smaller than three to five microns in largest dimension are flushed through the filtration medium, and the complex, if formed, is retained on the medium.
  • a color developing reagent including a substrate for the enzymatic colorimetric indicator label, is then deposited onto the filtration medium surface.
  • An indicator color is formed on the medium if the complex is present, thereby indicating the presence of organisms of the selected bacteria type in the sample.
  • a diagnostic kit for the determination of presence of organisms of a selected bacteria type fluid in accordance with this invention comprises (a) a first reagent including a bacteriophage capable of attaching to organisms of the selected bacteria type, a first member of a specific binding pair, preferably, biotin, being coupled to the bacteriophage, and (b) a second reagent including a second member of the specific binding pair, preferably avidin, coupled to a water-insoluble, water- suspendible solid phase carrier particle having a size approximating the size of such organisms.
  • a labeling agent is affixed to at least to the bacteriophage in the first reagent or to the carrier panicles in the second reagent, preferably to the carrier panicles in the second reagent.
  • the diagnostic kit may further comprise a filter medium which retains on it a complex of at least the organism, the bacteriophage and the solid phase, the complex being retained on the filter medium, if such organisms are present in the fluid tested, by contacting the fluid with the first and second reagents and depositing the resulting mixture onto the medium.
  • the diagnostic kit may further include a third reagent which tests for the presence of the labeling agent.
  • FIGS 1-4 are schematic or symbolic representations of the operation of this invention in successive stages or steps.
  • Figure 1 depicts a liquid sample in which labelled bacteriophage capable of attaching to the target organism under test are added to a liquid sample being tested for the presence of such organisms.
  • Figure 2 depicts the sample of Figure 1 after a water-insoluble, water- suspendible solid phase carrier particle is added to the liquid sample test.
  • Figure 3 schematically depicts the operation in which the water-insoluble complex of the target organism , the labelled bacteriophage and the carrier particle are separated from other particles, bacteria and bacteriophage.
  • Figure 4 schematically depicts a color producing reaction for detecting the complex.
  • Figure 5 schematically depicts a bacteriophage bound to the cell wall of a bacteria and coupled to a latex particle by means of avidin-biotin coupling agents.
  • the reference numeral 10 indicates a field of a sample being tested for the presence of cells 11 of organisms of a specific bacterial type.
  • this bacterial type is a Listeria organism, and cell 11 is labeled accordingly.
  • Reference numeral 12 refer to cells of non-Lwte ⁇ ' ⁇ -type bacteria, and, also, are accordingly labeled.
  • the bacterial cells have a typical one micron longitudinal dimension.
  • Reference numerals 13 refer to bacteriophage which have been added to sample 10. The bacteriophage 13 are capable of attaching to
  • bacteriophage 13 comprises a head 14, a tail 15, tail fibers 16, and a tail plate 17 viewable in the illustration. Tailplate 17 is shown attached to the cell wall of bacterial cell receptor site 24.
  • Bacteriophage 13 has affixed to its head 14, one of two members of a specific binding pair, in this embodiment, biotin, represented schematically and indicated by reference numeral 18. Bacteriophage 13 is illustrated in the drawings as having an approximate longitudinal dimension of 0.2 microns relative to the dimension of the bacterial cells.
  • FIG 5 there also is depicted a water-insoluble, water- suspendible solid phase carrier particle 19, suitably a latex sphere having a particle dimension approximating the longitudinal dimension of bacterial cell 11, in these drawings, a diameter of about one micron.
  • Figure 5 also schematically illustrates, coupled to latex sphere 19, the second member of the specific binding pair complementary to member 18.
  • the second member, in this embodiment, avidin is identified by reference numeral 20 as the schematic member 20.
  • the avidin member 20 is illustrated coupled
  • HRP horseradish peroxidase
  • the specific binding pair 18,20 and the conjugate 20,21 are not to any scale with reference to bacteriophage 13 or latex sphere 19. Only a single binding pair is illustrated but in practice the bacteriophage 13 is covered with biotin molecules and sphere l9 is covered with avidin/HRP conjugate.
  • bacteriophage 13 and water-insoluble, water- suspendible solid phase carrier panicles 19 are contacted with the samples 10.
  • Figure 2 depicts a field of sample 10 in which bacteriophage 13 and carrier panicles 19 have been contacted with the sample to form a test mixture, which includes miscellaneous food particles or the like 22.
  • non-Listeria cells 12, unattached bacteriophage 13. and solid carrier spheres 19 containing an avidin binding agent to which some bacteriophage 13 with a complimentary binding agent biotin have become attached depicted generally by reference numeral 23.
  • a water- insoluble complex 25 comprising at least the organism of interest 11. bacteriophage 13, and solid carrier particles 19.
  • a plurality of bacteriophage 13 are attached by base plate 17 to the cell wall receptor site of organism 11.
  • avidin/enzyme conjugates cover the surface of solid carrier particles 19. As illustrated in the case of one of them in Figure 5, these avidin/enzyme conjugates couple through the avidin member to the biotin binding pair member on the head of bacteriophage 13.
  • these biotin particles attached to the head of bacteriophage 14 and within stearic hinderance constraints, a plurality of the spheres 19 can bind with a single bacteriophage attached to cell 11.
  • the spheres bound to the phage attached to the target organism represent the primary layer of bound spheres.
  • the complex continues to enlarge as a result of chain like reaction of phage and spheres binding to the outer surfaces of the primary layer and thereby creating multiple outer layers.
  • Each of the spheres having a diameter of approximate dimension to that of the organism, a very large complex of a single bacteria, multiple bacteriophage, and multiple spheres is produced.
  • This complex or cluster has an approximate diameter which is substantially larger than the size of the organism under test.
  • a filtration medium indicated generally by reference numeral 27 is schematically shown in partial perspective section.
  • Filtration medium 27 includes a filtration surface 26 defining a plurality of filtration pores 28 in contact with an underlying absorbent bed 29 comprised of a wicking matrix of fibers.
  • this is accomplished by pouring a liquid sample 10 onto surface 26 of filtration medium 27.
  • the liquid is absorbed by the absorbent bed 29 through pores 28. Particles smaller than the diameter of pore 28 also pass through the pores into the absorbent bed where they are trapped.
  • a buffered washing solution is poured onto surface 26 after deposition of the sample 10 material containing the complex 25, to wash from surface 26 through pores 28 any remaining carrier particles 19, bacteriophage 13, non-specific bacterial cells 12. and other miscellaneous matter 22, leaving only the complex/cluster 25 atop the filtration medium surface.
  • the surface of the filtration medium 26 is tested to detect whether any complex 25 is present on the surface, as will be the case if specific organism under test
  • sample 10 (organism 11 in the drawings) was present in sample 10.
  • a substrate for the horseradish peroxidase affixed to the latex spheres has been poured onto the filtration surface.
  • the horseradish peroxidase enzyme acts on the substrate and yields a colored byproduct that colors the spheres and, also, the filtration surface. This produces a visual indication of color and positively indicates that the organism under test, cell 11, was present in sample 10.
  • Bacteriophage specific to a particular bacterial species are prepared and biotinylated by well known methods.
  • a particular bacteriophage is commercially available for the host bacteria.
  • Listeria monocytogenes is available as ATCC-23074 from the American Type Culture Collection, Rpckville, Maryland, and the bacteriophage specific to such bacterial host is available from the same source as ATCC-23074B1.
  • ATCC-23074B1 ATCC-23074B1
  • the following illustrates a procedure for preparation of a biotinylated bacteriophage reagent specific to Listeria monocytogenes, modified from Molecular Cloning (T. Maniatis, E.F. Fritsh and J. Sambrook; Cold Spring Harbor Laboratory, page 67 (1982)).
  • the materials employed are; tryptic soy digest broth
  • TLB titer containing 10 x 10** plaque forming units (PFU)/mL) as crude lysate or as cleaned preparation; and 1.0 mM MgSO
  • An aliquot of the overnight Listeria broth culture is mixed with bacteriophage (crude lysate or purified phage) in a ration of 10 parts bacterial culture, 1 part bacteriophage suspension. This should yield an initial phage titer in this suspension of 1 x 10 ⁇ PFU/mL. The suspension is mixed and incubated at 37 °C.
  • the absorbed bacteria:bacteriophage suspension is diluted 1:40 into tryptic soy broth supplemented with 10 mM MgSO The suspension is incubated at room temperature with rotary agitation (approximately 100 rpm) until the culture demonstrates clearing, indicating lysis. This can be a period from 6-18 hours. If the clearing phase is missed,
  • SUBSTITUTE SHEET the unreleased bacteriophage can be liberated from the host cells by the addition of chloroform to yield 1% of volume.
  • the chloroform treated suspension is incubated at room temperature for an additional 30 minutes with agitation to facilitate cell lysis.
  • the bacteriophage are collected from the lysed suspension by filtration through a glass fiber prefilter to remove large bacterial debris followed by filtration through a 0.45 ⁇ m membrane filter to yield bacteria free phage in the supematant. Cleared supernatants are stored at 4°C. in the crude state until further use or for long term storage. Long term storage of Listeria phage at 4°C. as crude lysate does not result in significant loss of titer for as long as one year. Greater titer losses occur after frozen storage. All crude bacteriophage preparations should be titered monthly.
  • phage lysate (titer should be at least 10 ⁇ PFU/ml); sterile flasks or bottles; sterile oakridge tubes for 10.000 g centrifugation; NaCl solid; DN'ase and Rn'ase (1 mg/ml in deionized H2O); ice bath; and Polyethylene Glycol 6000 (PEG6000).
  • Equipment used is a high speed centrifuge (Sorvall RC5B) with a SS34 angle head.
  • the supematant is collected into a fresh bottle or flask. Do not allow centrifuged material to remain in the tubes for long periods since the pellet will dislodge and return to solution.
  • solid PEG6000 is added to a final concentration of 10% (w/v). This is dissolved and incubated on ice for at least one hour. After incubation, this is centrifuged at 10,000 g for 10 minutes. Run centrifuge without brake and remove tubes immediately after termination of run. Supernatant is poured off and residual fluid is removed by standing the centrifuge tubes inverted on absorbent material for at least five minutes.
  • Precipitated bacteriophage is collected by gently washing the tubes with phosphate buffered saline to yield a final volume of 2% of the original lysate volume (e.g., original lysate - 1 liter; final lysate is collected in 20 ml of PBS).
  • the precipitate is easily collected using a short pasteur pipet. Each tube should be washed two times, therefore, volumes should be calculated accordingly.
  • the resuspended precipitate is finallv extracted with chloroform. Extracts are centrifuged at 5000 g for five minutes
  • SUBSTITUTE SHEET to separate the chloroform and aqueous phases.
  • the centrifuge should be run without braking and all tubes should be removed immediately at termination of the run.
  • the upper aqueous phase is collected with a short pasteur pipet, taking care not to disturb the dense white pellicle between the chloroform and aqueous phases.
  • the cleaned phage may be stored in glass vials at 4°C. for indefinite periods and should be assayed on a monthly basis to ascertain phage titer.
  • the bacteriophage are biotinylated, suitably as follows: (1) dilute phage to 10 ⁇ - lO 1 ⁇ PFU/ml in Carbonate/bicarbonate buffer; (2) prepare a 1 mg/ml solution of NHS-biotin in 1 ml of Dimethylformamide (DMF). This preparation may be stored at 4°C. and reused; (3) mix phage with NHS- biotin solution to yield approximately 2 x 10 ⁇ biotin molecules/phage.
  • DMF Dimethylformamide
  • Example: 10 ⁇ l of 1 mg/ml NHS-biotin/ 5 ml of reaction mixture approximately 3.4 x 10* 5 biotin molecules per ml; (4) Incubate at room temperature with gentle stirring for 1 hour; (5) dialyze versus three changes of 2 liters of PBS at 4°C. for approximately 36 hours; (6) collect dialysate, filter (0.45 ⁇ M) for sterility and titer; and (7) the biotinylated bacteriophage is stored at 4°C. until use.
  • a method of preparing a carrier particle reagent using polystyrene latex particles and conjugating the panicles with an avidin - horseradish peroxidase conjugate follows: 1. To a 3 mL screw top vial with a rubber cap liner add the following ingredients in the order listed: a. 1.0 mL of 0.05 M, Phosphate buffer, pH 7.3; b. 100 ⁇ L of Strepavidin/Horseradish Peroxidase Conjugate (TAGO, Inc. Burlingame, California, Catalog No. 6467); and c. 75 ⁇ L of l ⁇ m latex spheres (sulfate) (Interfacial Dynamics Corp.,
  • SUBSTITUTE SHEET 7 Aspirate the supernatant with a pasteur pipette and flexible tubing attached to a vacuum source. Add 4-6 mL of 0.05 M, Phosphate buffer, pH 7.3 to each tube and resuspend the latex.
  • AH reagents were stored in concentrated form at room temperature.
  • AH reagents were diluted 6-24 hours prior to assay in the following manner:
  • Phage Reagent No. 2:28 Dilute 1:400 in 0.01M phosphate buffered saline
  • Latex Reagent Dilute 1:1600 in PBS supplemented with 0.025% Tween 20; Bacterial cultures were incubated overnight in Listeria Enrichment Broth (DIFCO Laboratories, Inc., Detroit, Michigan) and then were diluted to 10 ⁇ CFU/ml (1 McFarland turbidity). This suspension was then diluted serially to 10 ⁇ CFU/ml in
  • the assay conditions used were as follows: (a) mix 100 ul of bacterial suspension (1()4 CFU/mL) with 100 ⁇ l of phage reagent, shake gently for 10 sec. and incubate at room temperature for three minutes: (b) add 100 ⁇ l of latex reagent, shake gently for 10 sec. and incubate at room temperature for two minutes; (c) pour mixture onto five micron (5 ⁇ m) nylon membrane and wash with PBS/Tween wash buffer; and (d) add substrate solution and wait three to five minutes for color to
  • Latex reagent batch number 1 was used for the first six months of the study and latex reagent batch number 2 was used from week 20 through week 76.
  • Example 2 Following the same test methodology and reagents and described in Example 1, the specificity of a Listeria specific biotinylated bacteriophage reagent employing the avidinylated horseradish peroxidase enzyme labeled latex particle reagent system of this invention was tested. Testing results is interpreted as follows: Blue-green color formation is considered positive, absence of color formation is considered negative.
  • Phage Reagent No. 2:28 Dilute 1:400 in 0.01M phosphate buffered saline
  • Latex Reagent Dilute 1:1600 in PBS supplemented with 0.025% Tween 20;
  • CFUs colony forming units
  • Phage Reagent No. 2:28 Dilute 1:400 in 0.01M phosphate buffered saline
  • Latex Reagent Dilute 1:1600 in PBS supplemented with 0.025% Tween 20; Bacterial cultures were incubated overnight in Listeria Enrichment Broth

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Abstract

Bactériophage spécifique d'une espèce ou d'un genre biotinylé(e) et suspension aqueuse de particules avidinylées en phase solide, marquées par un enzyme indicateur tel que la peroxydase du raifort, mis au contact d'un échantillon test et d'un complexe avec tout organisme des bactéries spécifiques à l'espèce ou au genre dans l'échantillon. Le complexe est séparé, de préférence par filtration, des particules de taille plus petite, et un réactif à coloration est appliqué au milieu du filtrage. La coloration révèle la présence dans l'échantillon testé d'une bactérie du type visé.
PCT/US1993/001627 1992-02-28 1993-02-26 Methode et necessaire a diagnostic pour la determination de bacteries WO1993017129A1 (fr)

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Cited By (19)

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Publication number Priority date Publication date Assignee Title
WO1998053100A1 (fr) * 1997-05-22 1998-11-26 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Procede base sur l'utilisation de bacteriophages pour la detection de molecules biologiques dans des echantillons biologiques
EP0940472A1 (fr) * 1996-09-27 1999-09-08 Organo Corporation Procede et appareil de detection de bacteries
WO2000031537A1 (fr) * 1998-11-23 2000-06-02 Carlsson Bjoern Detection de biomolecules a l'aide de tampons de phages
WO2001009370A2 (fr) * 1999-07-30 2001-02-08 Profos Ag Mise en evidence et identification de souches bacteriennes
WO2002061117A1 (fr) * 2001-02-01 2002-08-08 Profos Ag Detection et identification de groupes de bacteries
WO2003000888A2 (fr) * 2001-06-24 2003-01-03 Profos Ag Procede de separation de cellules bacteriennes et de composants cellulaires
US6524809B1 (en) 1998-06-04 2003-02-25 Microsens Biophage Limited Analytical method using multiple virus labelling
WO2004036177A2 (fr) * 2002-10-15 2004-04-29 Regents Of The University Of Minnesota Analyses permettant de detecter ou de quantifier des pathogenes et des contaminants bacteriens ou viraux
WO2005001475A3 (fr) * 2003-04-10 2005-03-03 Kent Voorhees Appareil et procede de detection d'organismes vivants microscopiques au moyen de bacteriophage
WO2006105504A1 (fr) * 2005-03-31 2006-10-05 Microphage Incorporated Appareil et methode de detection de micro-organismes a l'aide d'un bacteriophage marque
US7166425B2 (en) 2002-04-12 2007-01-23 Colorado School Of Mines Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells
WO2007075179A2 (fr) * 2005-02-18 2007-07-05 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanocomplexe bacteriophage/point quantique (phage-qd) destine a detecter des cibles biologiques dans des isolats cliniques et environnementaux
WO2008064241A2 (fr) * 2006-11-20 2008-05-29 Microphage Incorporated Procédé et appareil pour dosage diagnostique amélioré à base de bactériophages
ES2325825A1 (es) * 2008-03-17 2009-09-18 Consejo Superior De Investigaciones Cientificas (Csic) (75%) Procedimiento y sistema para dectectar y/o cuantificar bacteriofagos susceptibles de infectar una cepa huesped bacteriana predeterminada, uso de un dispositivo micrelectronico sensor para detectar dichos bacteriofagos y dispositivos microelectronico sensor para llevar a cabo dich.
US8216780B2 (en) 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
US8455186B2 (en) 2007-06-15 2013-06-04 MicroPhage™ Incorporated Method of detection of microorganisms with enhanced bacteriophage amplification
US8697434B2 (en) 2008-01-11 2014-04-15 Colorado School Of Mines Detection of phage amplification by SERS nanoparticles
US9441204B2 (en) 2008-04-03 2016-09-13 Colorado School Of Mines Compositions and methods for detecting Yersinia pestis bacteria
CN106093374A (zh) * 2016-06-03 2016-11-09 广州市进德生物科技有限公司 一种单组份tmb显色液及其制备方法

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EP0940472A1 (fr) * 1996-09-27 1999-09-08 Organo Corporation Procede et appareil de detection de bacteries
EP0940472A4 (fr) * 1996-09-27 2004-09-08 Organo Corp Procede et appareil de detection de bacteries
WO1998053100A1 (fr) * 1997-05-22 1998-11-26 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Procede base sur l'utilisation de bacteriophages pour la detection de molecules biologiques dans des echantillons biologiques
US6265169B1 (en) 1997-05-22 2001-07-24 Istituto Di Richerche Di Biologia Molecolare P. Angeletti S.P.A. Method based on the use of bacteriophages for the detection biological molecules in biological samples
US6524809B1 (en) 1998-06-04 2003-02-25 Microsens Biophage Limited Analytical method using multiple virus labelling
WO2000031537A1 (fr) * 1998-11-23 2000-06-02 Carlsson Bjoern Detection de biomolecules a l'aide de tampons de phages
US7087376B2 (en) 1999-07-30 2006-08-08 Profos Ag Detection and identification of bacterial strains
JP2003505105A (ja) * 1999-07-30 2003-02-12 プロフォス・アクチエンゲゼルシャフト 細菌株の検出および同定
WO2001009370A3 (fr) * 1999-07-30 2001-07-05 Profos Gmbh Mise en evidence et identification de souches bacteriennes
AU781090B2 (en) * 1999-07-30 2005-05-05 Biomerieux S.A. Detection and identification of bacterial strains
WO2001009370A2 (fr) * 1999-07-30 2001-02-08 Profos Ag Mise en evidence et identification de souches bacteriennes
WO2002061117A1 (fr) * 2001-02-01 2002-08-08 Profos Ag Detection et identification de groupes de bacteries
US8124355B2 (en) 2001-02-01 2012-02-28 Biomerieux S.A. Detection and identification of groups of bacteria
US8932580B2 (en) * 2001-06-24 2015-01-13 Hyglos Invest Gmbh Methods for purification of bacterial cells and cell components
WO2003000888A3 (fr) * 2001-06-24 2003-12-24 Profos Ag Procede de separation de cellules bacteriennes et de composants cellulaires
US7579142B2 (en) 2001-06-24 2009-08-25 Profos Ag Methods for purification of bacterial cells and components
WO2003000888A2 (fr) * 2001-06-24 2003-01-03 Profos Ag Procede de separation de cellules bacteriennes et de composants cellulaires
US20100047895A1 (en) * 2001-06-24 2010-02-25 Hyglos Invest Gmbh Methods for purification of bacterial cells and cell components
AU2002350489B2 (en) * 2001-06-24 2007-01-18 Biomerieux S.A. Method for separating bacterial cells and cell components
US8216780B2 (en) 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
US7166425B2 (en) 2002-04-12 2007-01-23 Colorado School Of Mines Method for detecting low concentrations of a target bacterium that uses phages to infect target bacterial cells
US7972773B2 (en) 2002-04-12 2011-07-05 Colorado School Of Mines Method for detecting concentrations of a target bacterium that uses phages to infect target bacterial cells
EP1556502A4 (fr) * 2002-10-15 2006-07-19 Univ Minnesota Analyses permettant de detecter ou de quantifier des pathogenes et des contaminants bacteriens ou viraux
EP1556502A2 (fr) * 2002-10-15 2005-07-27 Regents Of The University Of Minnesota Analyses permettant de detecter ou de quantifier des pathogenes et des contaminants bacteriens ou viraux
WO2004036177A3 (fr) * 2002-10-15 2005-02-24 Univ Minnesota Analyses permettant de detecter ou de quantifier des pathogenes et des contaminants bacteriens ou viraux
WO2004036177A2 (fr) * 2002-10-15 2004-04-29 Regents Of The University Of Minnesota Analyses permettant de detecter ou de quantifier des pathogenes et des contaminants bacteriens ou viraux
WO2005001475A3 (fr) * 2003-04-10 2005-03-03 Kent Voorhees Appareil et procede de detection d'organismes vivants microscopiques au moyen de bacteriophage
WO2007075179A3 (fr) * 2005-02-18 2007-10-18 Us Gov Health & Human Serv Nanocomplexe bacteriophage/point quantique (phage-qd) destine a detecter des cibles biologiques dans des isolats cliniques et environnementaux
WO2007075179A2 (fr) * 2005-02-18 2007-07-05 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanocomplexe bacteriophage/point quantique (phage-qd) destine a detecter des cibles biologiques dans des isolats cliniques et environnementaux
US8092990B2 (en) 2005-03-31 2012-01-10 Colorado School Of Mines Apparatus and method for detecting microscopic organisms using bacteriophage
WO2006105504A1 (fr) * 2005-03-31 2006-10-05 Microphage Incorporated Appareil et methode de detection de micro-organismes a l'aide d'un bacteriophage marque
WO2008064241A3 (fr) * 2006-11-20 2008-07-10 Microphage Inc Procédé et appareil pour dosage diagnostique amélioré à base de bactériophages
WO2008064241A2 (fr) * 2006-11-20 2008-05-29 Microphage Incorporated Procédé et appareil pour dosage diagnostique amélioré à base de bactériophages
US8455186B2 (en) 2007-06-15 2013-06-04 MicroPhage™ Incorporated Method of detection of microorganisms with enhanced bacteriophage amplification
US8697434B2 (en) 2008-01-11 2014-04-15 Colorado School Of Mines Detection of phage amplification by SERS nanoparticles
WO2009115633A1 (fr) * 2008-03-17 2009-09-24 Consejo Superior De Investigaciones Científicas (75%) Procédé et système de détection et/ou de quantification de bactériophages susceptibles d'infecter une souche hôte bactérienne prédéterminée, utilisation d'un dispositif capteur micro-électronique pour détecter lesdits bactériophages et dispositif capteur micro-électronique permettant de mettre en oeuvre ce procédé
ES2325825A1 (es) * 2008-03-17 2009-09-18 Consejo Superior De Investigaciones Cientificas (Csic) (75%) Procedimiento y sistema para dectectar y/o cuantificar bacteriofagos susceptibles de infectar una cepa huesped bacteriana predeterminada, uso de un dispositivo micrelectronico sensor para detectar dichos bacteriofagos y dispositivos microelectronico sensor para llevar a cabo dich.
US9441204B2 (en) 2008-04-03 2016-09-13 Colorado School Of Mines Compositions and methods for detecting Yersinia pestis bacteria
CN106093374A (zh) * 2016-06-03 2016-11-09 广州市进德生物科技有限公司 一种单组份tmb显色液及其制备方法

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