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WO1997047764A1 - Methode pour analyser des echantillons de matrice glucidique - Google Patents

Methode pour analyser des echantillons de matrice glucidique Download PDF

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Publication number
WO1997047764A1
WO1997047764A1 PCT/FI1997/000368 FI9700368W WO9747764A1 WO 1997047764 A1 WO1997047764 A1 WO 1997047764A1 FI 9700368 W FI9700368 W FI 9700368W WO 9747764 A1 WO9747764 A1 WO 9747764A1
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Prior art keywords
sample
enzyme
matrix
determined
carbohydrate
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PCT/FI1997/000368
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English (en)
Inventor
Jaakko Pere
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VTT Technical Research Centre of Finland Ltd
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VTT Technical Research Centre of Finland Ltd
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Publication of WO1997047764A1 publication Critical patent/WO1997047764A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • 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
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
    • G01N2400/26—Cellulose

Definitions

  • the present invention relates to a method according to the preamble of Claim 1 for treat ⁇ ing a biological sample containing a carbohydrate based matrix or collected into such a matrix such that the sample can be analysed in a more reliable way.
  • the sample is analysed in a way that as such is previously known in the art, for example, by growing a culture or by instrumental analysis, for at least one essential property of the material to be analysed.
  • This invention relates further to a method according to the preamble of Claim 20 for analysing biological material, and to a method according to the preamble of Claim 22 for assaying biological material containing a carbohydrate matrix.
  • the method of the invention may be applied to cases in which the sample to be analysed together with the sampling device or the sample as such is treated with an enzyme product.
  • the invention also relates to a test kit according to the preamble of the Claim 26.
  • pathogenic microbes proliferate, usually locally, in the body of a human being or an animal.
  • the bacterium that causes tonsillitis a group A streptococcus
  • forms bacterial growth on the mucous membranes of the pharynx can be assayed by taking a sample from the mucous membrane with a cotton stick or with a sampling device of some other material, and by growing a culture from the sample on a growth medium.
  • Streptococci like other microbes can be determined also by an analysis based on the detection of an antigen, such as latex agglutination, El A (enzyme immunoassay), immunofluorescence dyeing, chromatography, or RIA (radio immunoassay).
  • an antigen such as latex agglutination, El A (enzyme immunoassay), immunofluorescence dyeing, chromatography, or RIA (radio immunoassay).
  • an antigen such as latex agglutination, El A (enzyme immunoassay), immunofluorescence dyeing, chromatography, or RIA (radio immunoassay).
  • an antigen such as latex agglutination, El A (enzyme immunoassay), immunofluorescence dyeing, chromatography, or RIA (radio immunoassay).
  • Other biological materials may be determined with methods according to the basic technique described above, that is, by impregnating the fibre matrix of the sampling device with a
  • the object of the present invention is to remove the disadvantages of the prior art and to provide a novel solution for treating biological samples.
  • a particular object of the invention is to provide a method for enhancing the reliability of the analysis of biological samples.
  • the invention is based on the idea that a sample of biological material, which may contain a carbohydrate matrix or which has been collected or has been absorbed by a sampling device containing a carbohydrate matrix, is pretreated with a preparation containing hydrolytic enzyme activity before a more detailed analysis of the sample.
  • a pretreatment with a hydrolase particularly with an enzyme affecting the structural parts of the cell walls of plants, such as cellulase, hemicellulase, pectinase, amylase or a mixture thereof, may significantly improve the sensitivity and repeatability of the analyses of the samples.
  • the method for analysing biological material containing a carbohydrate-containing matrix and the test kit according to the invention are characterized by what has been set forth and described in the characterizing parts of Claims 22 and 26, respectively.
  • the invention may be applied to the treatment of a sample containing a carbohydrate-based matrix or one collected or absorbed by such a matrix.
  • a sample is also referred to with the term "carbohydrate- based (analyte) sample”.
  • Bio material in a broad sense is used to refer to any material that contains or has contained living organisms, their structural parts and/or decomposition products of their structures (for example, antigens) and/or substances secreted or produced by them and/or antibodies formed against them and/or to other analytes of biological origin.
  • biological material comprises body fluids and secretions often present in clinical analyses, such as blood, serum, plasma, liquor, ascites, pleural fluid, urine, excrement, pus, pharynx sample, saliva and sputa. The method may also be applied to the analysis of tissue samples.
  • materials consisting of carbohydrates or containing these are regarded as biological materials. These generally include plant materials and foodstuffs and fodder prepared from plant material. A particularly interesting embodiment involves excrement, which contains at least some polysaccharides and organic fibres depending on the diet.
  • the method may also be used in environmental investigations.
  • Significant embodiments of the invention in this area involve hygiene assays in the health services (hospitals, health centres) and the pharmaceutical, food and fodder industries. These embodiments comprise both microbiological quality control of the actual products and monitoring of the hygiene of working areas (including tables, benches, shelves, floors, walls, ceilings, drains and sewers) and process apparatus (such as machines, apparatus, pipe lines and air conditioning ducts). These determinations are performed to an increasing extent, for example, in the pharmaceutical industry and in the food and fodder industries (for example, in bakeries, dairies, prepared food production, abattoirs and fodder mixing plants) and in the forest industry.
  • the invention may also be useful when studying the indoor air quality.
  • Possible embodiments also comprise plant pathological analyses of, for example, grain, vegetables and fruit.
  • the pathogen virus, bacterium, fungus
  • the pathogen may be more easily extracted and isolated from the sample to be analysed.
  • the method is not limited to samples originating from or produced by living organisms; in principle any material which can be absorbed into a carbohydrate-based matrix or which contains such a matrix is suitable for analysis according to the present method.
  • carbohydrate-based matrix encompasses in the present context various structures comprising sugar polymers, which may be, for example, fibrous, layered or weblike.
  • Typical carbohydrate matrices include structures containing various plant fibres, such as cellulosic or lignocellulosic fibres (including cotton, viscose, ramie and linen fibres).
  • layers, typically produced by microbes, containing non-fibrous polysaccharides are found in many biological materials. Such layers include the plaque that covers teeth, phlegm, and mucus in pharynx, saliva and cervical samples, and biofilms formed of microorganisms and polysaccharide structures comprising their secretion.
  • sample refers to a representative part separated from the material to be analysed, from which the desired property of the material (the analyte) may be determined.
  • the sample may be in the carbohydrate- based matrix of the sampling device or on its surface or the sample itself may contain carbohydrate-based material. In the latter case, the sample as such is subjected to pretreatment according to the invention.
  • the pretreatment according to the invention comprises that before the determination of the desired property the sample and its carbohydrate matrix are subjected to an enzyme preparation that contains hydrolase activity that affects the carbohydrate structure in order to release the desired analyte from the carbohydrate matrix.
  • an enzyme preparation that contains hydrolase activity that affects the carbohydrate structure in order to release the desired analyte from the carbohydrate matrix.
  • the sample is brought into contact with such an amount of hydrolase enzyme that is sufficient to loosen the structure of the matrix, but which does not essentially dissolve the matrix. Partial dissolution of the matrix and subsequently formed degradation products may affect the analysis. Therefore, when the sample contains cellulosic fibres, it is generally attempted to break down less than 20 % of the fibre into mono- and oligosaccharides. This is achieved by using a suitable enzyme composition and suitable conditions for the treatment.
  • At least one preselected property is determined from the biological material.
  • This property may be the presence of some microorganism, such as a pathogenic or non-pathogenic bacterium, virus, yeast, fungus, protozoan, or a part or a product (for example, an enzyme) thereof, or it may be the presence or concentration of some molecule (for example, an antibody, a hormone, prion or other marker), compound or element (for example, a trace element).
  • microbes may be determined by growing a culture, by antigen determination or by determining the antibodies formed against them.
  • Hydrolase refers to an enzyme that unbinds ester linkages and glycosidic linkages of the carbohydrate structure. It is especially preferable to use such a hydrolase according to the present invention that affects the bonds between the monosaccharide units of the polysaccharide and/or the bonds between the side chains in the monosaccharide units and the monosaccharide.
  • the enzyme preparation containing hydrolase activity consists of a cellulase, hemicellulase, amylase, pectinase or a combination thereof
  • Suitable cellulases include cellobiohydrolases and endoglucanases; on the other hand, suitable hemicellulases include xylanases and mannanases.
  • the pectinase preparation used according to the invention principally affects the galacturonic acid, rhamnose, xylose, fructose, arabinose and/or galactose components, xyloglucan and/or arabinoglucan components of the cell walls in plant fibres.
  • pectin esterase polygalacturonase, exopolygalacturonase, pectin lyase, endoglucanase, endoxylanase and mannanase.
  • the enzyme product may contain mixtures of these, especially of cellulases and hemicellulases, but the product may also contain other enzymes in addition to hydrolases.
  • a sample containing a carbohydrate-based matrix or a biological sample collected or absorbed to a sampling device containing a carbohydrate based matrix is brought into contact with an enzyme, for example, by submerging the sample into an enzyme solution for a desired period of time.
  • the sample is shaken in the solution and/or it is allowed to stand in it.
  • the enzyme treatment is performed, for example, by mixing the sample with the enzyme solution whereafter the enzyme is allowed to react for a desired period of time before commencing the analysis.
  • the enzyme treatment is carried out at 0 - 60 °C, pH 3 - 10, preferably 3 - 8.
  • the time for the treatment may vary from 1 minute to about 48 hours.
  • the suitable amount and composition of the enzyme depend on the origin of the sample to be treated and on the sample matrix itself. In those cases where the sample is collected with a carbohydrate-based sampling device, a preferable dose of the enzyme is 0.02 - 5 mg/g sample. If the sample itself contains a carbohydrate-based matrix (for example, excrement, grain, fodder), a suitable enzyme dose is 0.5 - 20 mg/g sample.
  • a carbohydrate-based matrix for example, excrement, grain, fodder
  • a suitable enzyme composition is determined by the sample matrix: for a sample collected by a fibrous and cellulosic sampling device, a suitable enzyme preparation contains especially cellulase and pectinase.
  • An enzyme preparation suitable for a sample containing a carbohydrate-based matrix contains cellulase, pectinase and hemicellulase. If a sample containing large amounts of starch is subjected to the treatment (for example grain, fodder) it is suitable to have also amylase in addition to the enzymes mentioned above in the enzyme product.
  • the hydrolase treatment is preferably performed at room temperature, in a buffer solution with pH about 4 - 6, in which case the duration of the enzyme treatment is typically 10 - 60 min.
  • the buffer used may be a Na acetate solution, for example.
  • An embodiment in which the enzyme treatment is carried out especially at the storage temperature of the sample or at about 0 - 6 °C, is suitable for especially hygiene determinations. Also in this case it is most suitable to use a buffer solution as medium in order to stabilize the pH to the optimum pH value of the enzyme.
  • the time for treatment is 1 - 30 h.
  • the embodiment described herein above may be performed, for example, by placing a sample taken for a hygiene determination into an enzyme solution immediately after sampling and keeping it there overnight or until the sample has been transported to a laboratory for analysis.
  • a test kit according to the invention that is intended for sampling biological material contains sampling equipment which comprises a sampling device consisting of a carbohydrate-based fibrous material, with which a sample of biological material may be collected, and a pretreatment agent, with which the analyte in a fibrous a carbohydrate- based material may be released for determination. Further, the test kit may also contain equipment for analysis, with which the analyte can be determined, for example, by the instant diagnosis methods described below. The test kit may also contain only the pretreatment agent.
  • the pretreatment agent of the test kit contains an enzyme product containing hydrolase activity which releases the analyte to be determined from the carbohydrate-based matrix of the sampling device.
  • the enzyme product may contain in addition to an enzyme or enzymes a buffer that stabilizes the acidity (pH) of the solution, components preventing the growth of microbes and substances improving the stability of enzymes.
  • the enzyme concentration in the product is adjusted to such a value that a sufficient enzyme dose can be achieved when the product is used. In many cases a suitable enzyme concentration in the product is 0.05 - 0.5 % by weight.
  • a sampling pad or wad consisting of a cellulosic matrix, which is possibly attached to a holder suitable for sampling, is used as the sampling device.
  • the cellulosic matrix comprises cotton, viscose or cellulose mass or a chemically modified product prepared therefrom, such as viscose Also absorbent paper strips are suited for use as sampling devices
  • the microbes possibly contained in the sample may be determined by conventional methods based on growing microbe cultures, or by other diagnostic methods. Examples of the latter include antigen assay, which is based on recognizing structural parts typical for microbes by antibodies or other reagents that react specifically with these.
  • the usual methods for determining antigens include immunological methods, such as latex agglutination, EIA, RIA, FIA (fluorescence immuno assay), immuno fluorescence dyeing and immuno electrophoresis
  • the nucleic acid sequences characteristic of the microbes may also be determined by using labelled nucleic acid as a probe (nucleic acid hybridization method)
  • the conventional determination of microbes by growing cultures typically takes place in three stages, first the microbes possibly present in the sample are cultivated, then they are isolated into a pure culture and finally identified.
  • the sample may be cultivated by diluting onto a dish, whereby single bacte ⁇ a yield separate colonies which may be identified
  • enriching conditions may be used for cultivation, which causes only the microbe of interest to grow In the identifying step the methods described above may be used.
  • the method is especially preferably used for microorganisms of the genera Pseudomonas, Bacillus, Pediococcus, Candida, Streptococcus, Staphylococcus, Escherichia, Salmonella, Campylobacter or Chlamydia species and for some viruses, such as rota- and adenoviruses.
  • any microbe or virus that can be cultivated or otherwise identified is suitable for determination by to the method according to the invention.
  • sample treatment according to the invention unlike known extraction solutions, does not adversely affect the analyte in the sample. Because some soluble sugars, which may be used by microorganisms, for example, are released into the carbohydrate matrix due to the action of hydrolase, the determination of these by cultivation is facilitated.
  • the method may be used to significantly improve the analyzability of the sample; as has been described in the examples below, the sensitivity of analysis may increase manyfold.
  • FIG 1 a chromatogram of a commercially available cellulase product is shown and in figure 2 the decomposition of the structure of a cellulosic sampling pad as function of time with various enzyme dilutions is shown.
  • Cellulase refers to a preparation that contains one or more cellobiohydrolases (E.C. 3.2.1.91) and endoglucanases (E.C. 3.2.1.4).
  • E.C. 3.2.1.91 cellobiohydrolases
  • E.C. 3.2.1.4 endoglucanases
  • HEC hydroxyethyl cellulose 13 500 nkat/ml ⁇ -glucosidase 1 030 nkat/ml
  • FIG 1 a chromatogram (DEAE Sepharose, Pharmacia, phosphate buffer pH 7.2, elution with a salt gradient) of an Econase product is shown, which shows that the product contains all the main cellulases of Trichoderma fungus (CBH I, CBH II, EG I and EG II).
  • DEAE Sepharose Pharmacia, phosphate buffer pH 7.2, elution with a salt gradient
  • Pectinases are a large group of enzymes which can break down pectin matter in the cell walls of plants
  • the branched pectin matter is comprised of galacturonic acid, rhamnose, xylose, fucose, arabinose and galactose
  • xyloglucans and arabinoglucans through which the pectinous matter is attached to cellulose, are regarded as belonging to the pectin matter
  • Arabmogalactans bridge the protein and the pectin components in the plant cell wall
  • Pectin esterase, polygalacturonase, exopolygalacturonase and pectin lyase are examples of enzymes affecting pectin Hemicellulases refer to an enzyme preparation that contains xylanases, mannanases and enzymes affecting pectin matter.
  • Xylanases (E.C. 3.2.1.8) and mannanases (E.C. 3.2.1.78) were produced by a Trichoderma fungus and purified as has been described in the literature (Tenkanen et al. 1992, Stalbrand et al. 1993). These enzymes were added to a commercial pectinase product (Pectinex Ultra, Novo Nordisk, Denmark), which resulted in the following activities:
  • xylanase 7 150 nkat/ml mannanase 9 000 nkat/ml polygalacturonase 15 150 nkat/ml
  • the material to be examined was an Omni-SAL sampling pad for the collection of saliva samples, made from cellulosic material, and cut to small pieces for the experiment.
  • the pieces of sample were incubated for 2 - 6 hours at room temperature (18 - 23 °C) in a buffer solution (100 mM Na acetate buffer, pH 5), to which various amounts of cellulase were added (Econase, Primalco Ltd., Biotec, Rajamaki, Finland).
  • the enzyme dilutions were 1 : 1,000, 1 : 5,000 and 1 : 10,000.
  • the reference was a buffer solution without enzyme and Omni-SAL solution provided with the product. After incubation absorbance (A ,,,,,,,) at a wavelength of 600 nm was determined, which is a measure of the dissolution of the sampling pad into the buffer.
  • FIG 2 the breakdown of the structure of the sampling pad is shown as a function of time with different enzyme dilutions, and with the buffer and the Omni-SAL solution.
  • Pseudomonas fragi (Gram negative bacterium) Bacillus licheniformis (Gram positive bacterium) Pediococcus inopinatus (Gram positive bacterium) Candida utilis (yeast)
  • the growth medium had the following composition: 2.4 g of LAB-Lemco powder (Oxoid), 8 g of Nutrient Broth (Difco), 50 g of saccharose, 10 g of glucose and 10 g of fructose in 1000 ml of distilled water.
  • the growth medium (200 ml) was inoculated with 2 ml of a microbe suspension with a cell concentration of 10 8 -10 9 cfu/ml (cfu, Cole > forming units) and thereafter poured into growth tanks, where the steel plates were snaken (60 rpm) at +25 °C for 5 days. The growth medium was changed every other day by pouring the old medium away and by adding about 200 ml fresh growth medium. After cultivation the sample plates were rinsed twice with sterile water in the growth tanks. 13
  • the bacterial samples were collected from the steel plates with cotton sticks which had been dipped into water. After sample collection the cotton sticks were treated with the following enzyme solutions.
  • cellulase (dilutions 1 : 100 and 1 : 500)
  • pectinase (dilutions 1 :100 and 1:500) mixture of cellulase and pectinase (1:1, diluted to 1:100 and 1:500)
  • the enzyme treatments were performed in a buffer (50 mM Na acetate buffer, pH 5) at room temperature (18 - 23 °C) for one (1) hour.
  • a corresponding treatment (buffer) without enzyme was used as the reference.
  • the sample tubes were mixed at the beginning and at the end (30 s). After the treatment the sample was serially diluted into a physiological salt solution containing peptone, and from suitable dilutions transfers were made onto nutrient agar plates in two replicates, which were grown at +30 °C for 2 days.
  • Organism Detached colonies, log cfu/cm 2 , after sample treatment with cellulase pectinase mixture buffer of pectinase and cellulase
  • the enzyme treatment improved cultivation yield of the microbes, which were collected from a biofilm sample using a cotton stick Depending on the composition of the biofilm either cellulase alone or a mixture of cellulase and pectinase gave the best results
  • the test used was a Tandem ICON Strep A instant test kit, the sampling devices were a cotton stick and a dacron stick provided with the kit Artificial samples were prepared by taking a pharynx sample with a cotton or dacron stick (healthy pharynx) according to the instructions and thereafter inoculating the sticks with 50 ⁇ l of a Streptococcus pyogenes strain grown over night at a concentration of 10 6 or 10 5 bacteria/ml The sampling sticks were submerged for 5 seconds into the enzyme solution and were placed in a sterile tube for 30 minutes or 1 hour.
  • the enzyme solutions contained cellulase, hemicellulase or a mixture of cellulase and hemicellulase. Before the treatment the enzymes were diluted 1:10 into physiological NaCl After the enzyme treatment an instant test was performed according to the instructions Colour intensity was determined visually
  • a Wellcozyme Chlamydia Specimen Collection Kit (female) cotton stick was used as the sampling device
  • the Chlamydia antigen was determined with an EIA method (Wellcozyme Chlamydia Murex)
  • the antigen ("sample") was an impure Chlamydia preparation (Orion Diagnostica, Espoo, Finland) diluted in PBS
  • the enzyme solution (cellulase) was diluted (1/100 and 1/500) into dilute HC1 with at pH 5.0.
  • the enzyme treatments were made with the following enzyme preparations: cellulase, hemicellulase (xylanase + mannanase + pectinase) and a mixture of cellulase and hemicellulase.
  • the enzyme solutions were diluted 1 : 10 into a physiological NaCl solution.
  • the excrement sample (1 g) was mixed (in a Vortex mixer for 1 minute) into 9.0 ml of the enzyme solution. Thereafter, the samples were allowed to stand at room temperature for various times (30, 60, 120 and 480 minutes), whereafter the sample absorbances were measured at 600 nm. Before each measurement the instrument was set to zero with a reference sample ( 1 g excrement without enzyme) and then with the enzyme solution (coloured). The results are presented in Table 4.
  • Hemicellulase 1.25 1.60 Mixture of cellulase and hemicellulase 3.00 3.20

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Abstract

L'invention concerne une méthode permettant de traiter un prélèvement biologique recueilli dans ou contenant une matrice à base de glucides, de façon à pouvoir analyser ledit prélèvement d'une manière plus fiable. Selon l'invention, on met le prélèvement en contact avec un produit enzymatique ayant une activité cellulase, hémicellulase, amylase et/ou pectinase, afin de relâcher la structure de la matrice. L'invention concerne également une méthode permettant d'analyser des prélèvements biologiques et une trousse d'analyse permettant de prélever des échantillons dudit matériau. La trousse comprend typiquement un dispositif de prélèvement, qui comporte une partie constituée d'un matériau fibreux permettant de recueillir ou d'absorber le matériau biologique, et un agent de prétraitement ayant une activité hydrolase, qui permet de libérer le matériau du prélèvement absorbé dans le matériau fibreux afin de l'analyser.
PCT/FI1997/000368 1996-06-11 1997-06-11 Methode pour analyser des echantillons de matrice glucidique Ceased WO1997047764A1 (fr)

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FI962408A FI101809B (fi) 1996-06-11 1996-06-11 Menetelmä näytteen analysoimiseksi hiilihydraattimatriisista
FI962408 1996-06-11

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001090297A1 (fr) * 2000-05-25 2001-11-29 Cellomeda Oy Dispositif de recueil d'echantillons destine au diagnostic de maladies et utilisation de ce dernier
WO2001079527A3 (fr) * 2000-04-18 2002-08-15 Pearl Technology Holdings Llc Verification de la fidelite sexuelle et d'un crime sexuel
WO2010043668A1 (fr) * 2008-10-17 2010-04-22 Zentech S.A. Points de sang séché pour une analyse de sang
WO2016071805A1 (fr) * 2014-11-07 2016-05-12 Stora Enso Oyj Procédé amélioré pour la détermination de micro-organismes
US10520485B2 (en) 2013-04-12 2019-12-31 Upm-Kymmene Corporation Analytical method for determining the concentration of oxidized nanofibrillar cellulose in a sample

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0153477A1 (fr) * 1984-02-27 1985-09-04 Becton Dickinson and Company Test diagnostique pour le streptocoque A
US5432097A (en) * 1993-11-09 1995-07-11 Yourno; Joseph Method for recovery of blood cells from dried blood spots on filter paper
US5466579A (en) * 1987-12-28 1995-11-14 Psychemedics Corporation Hair analysis method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0153477A1 (fr) * 1984-02-27 1985-09-04 Becton Dickinson and Company Test diagnostique pour le streptocoque A
US5466579A (en) * 1987-12-28 1995-11-14 Psychemedics Corporation Hair analysis method
US5432097A (en) * 1993-11-09 1995-07-11 Yourno; Joseph Method for recovery of blood cells from dried blood spots on filter paper

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079527A3 (fr) * 2000-04-18 2002-08-15 Pearl Technology Holdings Llc Verification de la fidelite sexuelle et d'un crime sexuel
WO2001090297A1 (fr) * 2000-05-25 2001-11-29 Cellomeda Oy Dispositif de recueil d'echantillons destine au diagnostic de maladies et utilisation de ce dernier
WO2010043668A1 (fr) * 2008-10-17 2010-04-22 Zentech S.A. Points de sang séché pour une analyse de sang
US10520485B2 (en) 2013-04-12 2019-12-31 Upm-Kymmene Corporation Analytical method for determining the concentration of oxidized nanofibrillar cellulose in a sample
WO2016071805A1 (fr) * 2014-11-07 2016-05-12 Stora Enso Oyj Procédé amélioré pour la détermination de micro-organismes
CN107027314A (zh) * 2014-11-07 2017-08-08 斯道拉恩索公司 测定微生物的改进的方法
JP2017532977A (ja) * 2014-11-07 2017-11-09 ストラ エンソ オーワイジェイ 微生物の決定のための改善された方法
EP3215632A4 (fr) * 2014-11-07 2018-06-20 Stora Enso Oyj Procédé amélioré pour la détermination de micro-organismes
US10017806B2 (en) 2014-11-07 2018-07-10 Stora Enso Oyj Method for determination of microorganisms
CN107027314B (zh) * 2014-11-07 2021-02-19 斯道拉恩索公司 测定微生物的改进的方法

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