EP0298094A1 - Procede d'identification salmonella - Google Patents

Procede d'identification salmonella

Info

Publication number
EP0298094A1
EP0298094A1 EP19880900198 EP88900198A EP0298094A1 EP 0298094 A1 EP0298094 A1 EP 0298094A1 EP 19880900198 EP19880900198 EP 19880900198 EP 88900198 A EP88900198 A EP 88900198A EP 0298094 A1 EP0298094 A1 EP 0298094A1
Authority
EP
European Patent Office
Prior art keywords
bacteriophage
specific
virus
organism
peroxidase
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Withdrawn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
EP19880900198
Other languages
German (de)
English (en)
Inventor
William J. Hubbard
Jon S. Lane
Raymond E. O'bear
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.)
McDonnell Douglas Corp
Original Assignee
McDonnell Douglas Corp
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 McDonnell Douglas Corp filed Critical McDonnell Douglas Corp
Publication of EP0298094A1 publication Critical patent/EP0298094A1/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Withdrawn legal-status Critical Current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
    • C12Q1/02—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
    • C12Q1/02—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
    • C12Q1/10—Enterobacteria
    • 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

  • Applicants have discovered and perfected a new test method for directly testing for the presence of unknown organisms.
  • Applicants method is fast acting and produces a test result which has a high degree of certainty.
  • Applicants test procedure provides qualitative and quantitative evaluation of unknown organisms.
  • the test may be used to identify a particular species, type or genus.
  • Applicants method uses a specific agent which rapidly attaches to a target test organism.
  • the agent is labeled, for positive identification.
  • Applicant's method may use a variety of conventional labeling or reporting techniques.
  • the agent may be marked or tagged for identification using radioactive or chemical marking, for example, peroxidase labeling, fluorescent labeling, biotin labeling or other identification for the agent.
  • the marked or tagged agent is then brought into contact with a test unknown organism.
  • agent attaches to the organism its presence can be determined by detecting the tag or marker to provide a positive identification for that particular organism.
  • agent is a bacteriophage. It is generally accepted that bacteriophages are narrowly restricted in their range of host bacteria. Some bacteriophages are specific for a particular species of bacteria. However, there are also some bacteriophages which are specific to particular genuses.
  • bacteriophages may be labeled or tagged, as described herein, and can then be used to identify a bacterium, based on the binding or non-binding to that particular bacteria species, type, or genus. Since a bacteriophage is smaller than the organism to which it binds, free bacteriophage can be separated from bacteria by filtration through an appropriately sized medium, for example, inert filter paper. The filter traps the bacteria and permits the unbound smaller bacteriophage to pass through the filter. However, if the bacteria is the one to which the bacteriophage is specific, the bacteriophage will bind to the bacteria within a very short time.
  • the bacteria will be trapped on the filter with the bacteriophage attached. If the bacteriophage is labeled its presence can be determined by developing the tag or marker and the presence of that specific bacteria to which the particular bacteriophage attaches can then be determined. Of course, the greater the number of the specific bacterial organisms present the greater the number of bacteriophage which will be found attached to the specimen on the filter, yielding a greater signal from the reporter groups.
  • FIG. 1 is a curve showing the rate of biphenol consumption as a function of hydrogen peroxide concentration
  • FIG. 2 is a curve showing the rate of biphenol consumption as a function of biphenol concentration
  • FIG. 3 illustrates the limit of sensitivity of liquid assays for peroxidase using biphenol/H 2 0 2 .
  • FIG. 4 illustrates the stability of biphenol in the presence of H-0 2 compared to other systems.
  • Bacteriophage are easy to acquire. Suitable wild bacteriophage may be harveste ⁇ from nature, isolated very readily by conventional means and grown to substantial quantity on the specific host bacterium. Applicants have harvested and isolated bacteriophage specific for Salmonella, E. coli, Staphylococcus aureus and Pseudomonas, among others. In addition, common bacteriophage, for example, Tl, T2, T3, T4 and lambda, which are specific for E. coli, are readily available, as are other known bacteriophage.
  • ATCC 15693-B1 is specific for Pseudomonas aerugenosa
  • ATCC 6538P is specific for Staphylococcus aureus
  • ATCC 6051-B1 is specific for Bacillus subtilus
  • ATCC 23074-B1 is specific for Listeria monocy ogenes.
  • Naturally available and collected bacteriophage are specific for Ca pylobacte , Mycobacterium tuberculosis and others.
  • Bacteriophages rapidly bond to the outside of host bacteria and, though infecting .the bacteria, leave the shell of the bacteriophage present on the surface of the host.
  • the properties of bacteriophages in binding to a host are noted in Reco binant DNA, A Short Course, Watson et al. pages 14, 15, 23 and 24, W.H. Freeman & Company, New York 1983 and have been noted in "The Mechanism of Virus Attachment to Host Cells. IV. Physiochemical Studies on Virus and Cell Surface Groups," Arch. Biochem. Biophys. Vol. 51 (1954), Puck et al. , pages 229 through 245.
  • Bacteriophage are particularly advantageous as a test reagent in that relative to its host bacterium, and to other carriers such as antibodies or gene probes, the bacteriophage is relatively large, enabling the tester to bind sizeable quantities of tags or marker elements, such as, enzyme markers, fluorescent markers, or radioactive markers, to the individual bacteriophage.
  • the protein shell of the bacteriophage remains outside and attached to the bacterium after infecting the host bacterium so that the presence of the bacteriophage can be readily identified.
  • the bacteriophage is also sufficiently smaller than the host bacteria to permit the unattached bacteriophage to be readily separated from the bacteria in a test specimen by filtration.
  • bacteriophages to provide a marker that identifies the presence of the bacteriophage or the bacteriophage bound to a host can be readily accomplished.
  • the shell of bacteriophage is proteinaceous and a variety of markers can be bound to the bacteriophage surface by using a protein linker.
  • the enzyme horseradish peroxidase (HRP) may be linked to a the protein on the shell of a bacteriophage with a bifunctional cross linking reagent, dimethyl suberimidate 2HC1, an imidoester.
  • HRP horseradish peroxidase
  • bacteriophages-linker-peroxidase complex Other enzymes may also be used, for example, alkaline phosphatase and beta-galactosidase may also be used as well as the use of other bifunctional cross linkers such as other amino reacting cross linkers and thiol reacting cross linkers.
  • Fluorescent labels may be attached by using a fluorescent label-fluorescene isothiocyanate through the epsilon-amino groups of lysine found in the bacteriophage capsomeres. Other fluorescent molecules such as rhodamine may also be used in a similar manner. Labeling the bacteriophages with biotin type reporter groups using a succini ide ester group may also be used.
  • Radioactive labeling of the bacteriophages with various radioactive compounds could also be used, for example, radioactive iodine 125 may be linked to the protein of the bacteriophage shell by a conventional reaction.
  • a modified Nakane method of binding horseradish peroxidase may be used to label bacterophage.
  • the HRP-CH-. is buffered with 0.30M NaHCO- and separated by chromotography on a 0.5m exclusion column.
  • the HRP-CH 3 is then oxidized with 0.04M NalO, to form HRP-CHO (horseradish peroxidase aldehyde).
  • HRP-CHO is buffered to pH 9.0 with a a 2 C0 3 buffer and separated on a 0.5m exclusion column to produce the activated HRP enzyme.
  • the bacteriophage is grown on a host bacterium culture, for example S. typhimurlum. The lysed bacteriophage is filtered through a 0.45 micron filter and is separated from the culture material. The activated enzyme is added to the bacteriophage and reacted for 4 to 6 hours at room temperature.
  • the tagged bacteriophage is buffered to pH 7.0 with a 50mM PO. buffer and separated on a Biogel A 0.5m exclusion column. The separated tagged bacteriophage is then assayed and adjusted for titer.
  • Salmonella Species Type Test Salmonella specific bacteriophage was harvested from nature and isolated on a host culture of Salmonella typhimurium.
  • Salmonella Group 1 worthington Positive anatu Positive cholerae var. suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive
  • Salmonella Group 4 flint Positive marina Positive Salmonella, Group 5 brookfield Positive bongar Positive
  • test procedure for each organism listed in the Table I was as follows: A culture of the organism was slurried to a concentration of 10 organisms per ml. A 100 ul sample of the organism slurry was combined with 50 ul of the labeled bacteriophage reagent. The reagent was standardized to a titer
  • a test for reliability of the labeled bacteriophage was conducted on the array of bacteria shown in Table II and Table III.
  • the bacteriophage used were obtained from the American Type Culture Collection, with the exception of applicants' Salumonella bacteriophage. The bacteriophage were specific for the particular organism, as noted herein. Each bacteriophage used was labeled with horseradish peroxidase using the modified Nakane method described. The tests were conducted as described in Example I and developed using para-para-biphenol. Table II
  • Applicants have further found that they can produce and isolate a horseradish peroxidase having a high level of polymerization and which is readily coupled with bacteriophage.
  • Applicants' coupled enzyme and bacteriophage may have an effective ratio of enzyme to bacteriophage of above about 200 enzyme units per bacteriophage and preferably above about 1000 enzyme units per bacteriophage.
  • the assay described herein has a sensitivity of detecting fewer than 10,000 bacteria per assay test well, including as few as 100 bacteria per assay well.
  • periodate oxidation is particularly useful for coupling peroxidase, especially when used with methods, such as column chromotography, for obtaining polymeric enzyme activity from the HRP.
  • the basic periodate oxidation is described in "Practice and Theory of Enzyme Immunoassays," by P. Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology, v. 15, R.H. Burdon and P.H. van Krippenberg (eds) Elsevier, N.Y. 1985.
  • a suitable active polymeric fraction of HRP may be obtained by column chromatography over BioGel A 0.5m.
  • the preparations that yield the best activity are those fractions between the void and the leading edge of the major enzyme containing peak.
  • Other methods of increasing the enzyme count per bacteriophage may be used. These methods include but are not limited to the addition of compounds such as bifunctional cross-linking agents, divalent or multivalent amlne contaning compounds, divalent or multivalent aldehyde containing compounds, lecithins, and anti-enzyme antibodies.
  • Those skilled in the art may also appreciate that the sensitivity of the enzyme tagged bacteriophage assay depends on the number of active enzymes per phage, and that the requisite ratio of enzymes per bacteriophage may be generated by methods not requiring enzyme polymers. These methods include but are not limited to the successive or in situ chemical layering of monomeric enzymes onto the bacteriophage surface, or by prior coupling of a polymeric or particulate matrix that can accommodate the requisite number of enzymes per bacteriophage.
  • Various methods may be used to separate bacteria bound reagent from free reagent.
  • One such method practiced by the applicants is centrifugation and washing of the pellet in buffer. One or two washes are sufficient to reduce nonspecific background to an acceptable level.
  • the preferred . method for separating free enzyme-tagged bacteriophage from bacteria bound enzyme-tagged bacteriophage is by filtration on a filter matrix that retains bacteria but passes enzyme-tagged bacteriophage, followed by washing with a detergent containing buffer. Low protein binding filters used for bacterial sterilization of fluids having pores of 0.45um give satisfactory results.
  • the filter-trapped bacteria/enzyme-tagged bacteriphage complex is then contacted with a solution of chromogenic substrate.
  • the substrate is capable of undergoing enzymatic transformation upon contacting the enzyme-tagged bacteriophage to a chromophoric product.
  • soluble chromophoric products may serve as positive indicators of target bacteria, the applicants have found that a chromophoric product that binds to or otherwise is trapped by the filter matrix upon filtration of the reaction mixture gives increased sensitivity.
  • the preferred chromogenic substrate is para-para-biphenol, as described herein.
  • a filter based assay of this type has a sensitivity of detecting less than 10,000 bacteria per assay well, and with reagents having greater than 2,000 enzymes per bacteriophage, less than 100 bacteria per assay well.
  • React horseradish peroxidase (Sigma, Type I) with 0.032M formaldehyde in 0.3M NaHC0 3 buffer, pH 8.1 for 30 minutes. Reduce the Schiff's base residues with either lmg NaBH,/mg protein or 0.15 mg NaCNBH,/mg protein. Incubate at 4°C for 16 to 24 hours. Chromatograph the reaction mixture on a BioGel A 0.5m size exclusion column that had been equilibrated in 0.3 M bicarbonate buffer pH 8.1. Pool all fractions excluding aggregates in the void volume and low molecular weight reactants in the totally included peak. Oxidize the pooled fractions with 0.04M NalO, for 30 minutes at room temperature.
  • the typical pooled concentrate has an absorbance at 280nm of between 0.5 to 0.8, a 403 nm/280 nm absorbance ratio of between 0.3 to 0.8, and contains between 0.44 to 2.6 uM HRP by the pyrogallol assay.
  • Example IV Coupling Activated HRP to Salmonella Specific Bacteriophage
  • a Salmonella specific bacteriophage was harvested from raw sewage and isolated on a host culture of Salmonella typhimurium. (A culture of this bacteriophage, designated ATCC
  • the final concentration of phage in the reaction is between 10 8 to 109 pfu/ml, and the final concentration of enzyme is 0.11 to 0.6 uM.
  • the enzyme readily binds to the protein coating of the bacteriophage, probably by attaching to lysine residues present in the protein coating.
  • HRP Salmonella Species Type Test
  • Example 2 Test the enzyme tagged bacteriophage of Example 2 against representative Salmonella species and serologically closely related species of Citrobacter as shown in Table II.
  • the test procedure for each organism listed in Table II was as follows: Slurry a culture of test organism to a concentration of 10 organisms per ml. Add- a- 100 ul sample of each organism slurry to a filter well of a BioRad microfiltration apparatus having a 0.45 micron HT Tuffryn filter. Remove the solution by vacuum filtration in the apparatus. Add a 50ul aliquot of
  • Salmonella Group 1 worthington Positive anatum Positive cholerae var . suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive
  • Salmonella species type test to be 100 organisms/ml.
  • a particularly useful substrate for detecting the presence of enzyme coupled peroxidase is one incorporating para-para-biphenol.
  • a substrate of this type is use in Examples I and V above, for example. This substrate has been found to be highly sensitive for colorimetric analysis and is highly stable.
  • the problem in detecting peroxidase activity is that high sensitivity is linked to high background.
  • the problem is further complicated by the need for soluble substrate that becomes insoluble after being acted upon by the enzyme.
  • the ideal substrate should be (1) rapid, (2) soluble to the extent that it saturates the enzyme, (3) stable to oxidation by hydrogen peroxide in the absence of enzyme, (4) converted to a colored, highly insoluble product by the action of peroxidase.
  • 4,4'-biphenol fullfills the four criteria enumerated above. (1) It exhibits a rate constant M—1 sec—1) as high or higher 6 than any other substrate known. (2) The overall rate of reaction is independent of biphenol
  • Solid 4 '-biphenol is added (lg/L) to aqueous buffer
  • Solid material is removed by filtration (0.45u filter) and hydrogen peroxide is added (10-lOOul of 30% H 2 O 2 /100ml biphenol solution) .
  • the solution is ready to use.
  • the substrate is added to the surface of a filter upon whih peroxidase has been deposited. After 5-60 minutes, the solution is filtered.
  • peroxidase levels as low as 1 fmol (femptomole, 10 -15 moles) can be detected. No color change can be detected in the absence of enzyme.
  • This substrate can be used in filter assays for detecting peroxidase, peroxidase labeled reagents (antibodies, gene probes, bacterial viruses and the like), and coupled enzyme assays where the final enzyme is peroxidase.
  • peroxidase labeled reagents antibodies, gene probes, bacterial viruses and the like
  • coupled enzyme assays where the final enzyme is peroxidase.
  • H 2 0 2 in the working solution was an average of determinations made at 240 nm (Hildebrant, A.G. and I. Roots,
  • Solid Phase Assay A 96 well dot-blot apparatus (BioRad) with a 0.45um pore size polysulfone membrane (HT-Tuffryn, Gelman) was used to perform reactions and then trap insoluble product. Results were scored visually, based upon the appearance of any brown material apparent after filtration. The effect of pH, ionic strength, hydrogen peroxide concentration, incubation times and test sensitivity were tested.
  • Buffer 46.9mM phosophate, pH7.0
  • Rate -.k 4 [Peroxidase! rH202> k 1 [H 2 0 2 ] + k 4 [Biphenol]
  • Buffer 46.9mM phophate, pH 7.0
  • Buffer 37.5mM phophate, pH 7.0
  • Buffer 0.0375M Phosphate, pH 7.0
  • Buffer 0.1 MNaH 2 PO4,pH6.0
  • MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloric acid salt); 0.14mg/mL plus (3-dimethylamino benzoic acid); 5mg/mL
  • PYROGALLOL (1,2,3-trihydroxy benzene); 50mg/mL
  • H-Y Hanker-Yates reagent Polysciences; a combination of p-phenylenediamine and pyrocatechol); 0.5mg/mL BIPHENOL a saturated solution, 1.5 x 10 -4M.
  • Blot diluton assay P.aeruginosa ' phage reagent 1. Prepare a series of microorganism concentrations of the host microorganism used to propagate the phage. The solutions should be a series of 11 1:2 dilutions in 0.45%
  • Example IX Determination of peroxidase concentration in an unknown sample.

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Abstract

Test direct d'identification des organismes bactériels faisant appel à un bactériophage spécifique qui se combine avec les organismes cibles. Le marquage du bactériophage permet d'identifier réellement l'espèce ou le genre de la bactérie. Les marqueurs peuvent être radioactifs, fluorescents ou enzymatiques. On peut recourir au biphénol comme substrat pour les marqueurs utilisant la peroxydase.
EP19880900198 1986-12-01 1987-12-01 Procede d'identification salmonella Withdrawn EP0298094A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93651586A 1986-12-01 1986-12-01
US936515 1986-12-01

Publications (1)

Publication Number Publication Date
EP0298094A1 true EP0298094A1 (fr) 1989-01-11

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ID=25468751

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Application Number Title Priority Date Filing Date
EP19880900198 Withdrawn EP0298094A1 (fr) 1986-12-01 1987-12-01 Procede d'identification salmonella

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EP (1) EP0298094A1 (fr)
AU (1) AU1043788A (fr)
CA (1) CA1313111C (fr)
WO (1) WO1988004326A1 (fr)

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EP0439354A3 (en) * 1990-01-24 1992-06-17 Amoco Corporation Signal generating moiety and method for use
US5168037A (en) * 1990-08-02 1992-12-01 Phyllis Entis Method for the preparation of a labelled virus without the inactivation of viral binding sites and method of assay utilizing said labelled virus
BE1006312A3 (fr) * 1991-11-29 1994-07-19 Univ Catholique Louvain Procede de selection de microorganismes recombinants comportant a leur surface au moins une molecule a activite enzymatique.
AU3776493A (en) * 1992-02-28 1993-09-13 Paul W. Judkins Method and diagnostic kit for determination of bacteria
DK63093D0 (da) * 1993-06-02 1993-06-02 Foss Electric As Improved method
JP3615810B2 (ja) * 1994-12-05 2005-02-02 東北電子産業株式会社 細菌の検出方法及び検出装置
JP3270722B2 (ja) * 1996-09-27 2002-04-02 オルガノ株式会社 細菌の検出方法及び検出装置
AU7113398A (en) * 1997-04-24 1998-11-13 Idaho Research Foundation Inc., The Phages, methods for growing and detecting them and their use
US8216780B2 (en) 2002-04-12 2012-07-10 Microphage (Tm) Incorporated Method for enhanced sensitivity in bacteriophage-based diagnostic assays
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
WO2006105414A2 (fr) * 2005-03-31 2006-10-05 Colorado School Of Mines Appareil et procede de detection d'organismes microscopiques au moyen d'un microphage
WO2007035504A1 (fr) * 2005-09-15 2007-03-29 Microphage Incorporated Methode et appareil d'identification de microorganismes basee sur des bacteriophages
WO2008064241A2 (fr) * 2006-11-20 2008-05-29 Microphage Incorporated Procédé et appareil pour dosage diagnostique amélioré à base de bactériophages
AU2008265989B8 (en) 2007-06-15 2012-01-12 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
CN105823876B (zh) * 2016-03-18 2018-01-16 南昌大学 一种针对沙门氏菌的检测方法
US20190078133A1 (en) * 2017-09-08 2019-03-14 The Charles Stark Draper Laboratory, Inc. Detection and identification of bacteria and determination of antibiotic susceptibility using bacteriophage and reporter molecules

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AU2780284A (en) * 1983-05-16 1985-11-21 Packard Instrument Co. Inc. Method of measuring atp and concentrating and measuring unicellular organisms
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GB2181542A (en) * 1984-03-19 1987-04-23 Marius Constantin Teodorescu Bacteriophages as recognition and identification agents
CA1277931C (fr) * 1984-06-05 1990-12-18 Shimon Ulitzur Detection et (ou) identification de microorganismes dans les echantillons a l'aide de la bioluminescence et d'autres marqueurs exogenes introduits par manipulation genetique
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CA1313111C (fr) 1993-01-26
WO1988004326A1 (fr) 1988-06-16
AU1043788A (en) 1988-06-30

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