WO1997047764A1 - Method for analysing samples from a carbohydrate matrix - Google Patents

Method for analysing samples from a carbohydrate matrix Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
sample
enzyme
matrix
determined
carbohydrate
Prior art date
Application number
PCT/FI1997/000368
Other languages
French (fr)
Inventor
Jaakko Pere
Original Assignee
Valtion Teknillinen Tutkimuskeskus
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 Valtion Teknillinen Tutkimuskeskus filed Critical Valtion Teknillinen Tutkimuskeskus
Publication of WO1997047764A1 publication Critical patent/WO1997047764A1/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/924Hydrolases (3) acting on glycosyl compounds (3.2)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24Homoglycans, 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
    • G01N2400/26Cellulose

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

Abstract

The invention relates to a method for treating a biological sample collected in or comprising a carbohydrate based matrix such that the sample may be analysed more reliably. According to the invention the sample is brought into contact with an enzyme product comprising cellulase, hemicellulase, amylase and/or pectinase activity in order to loosen the matrix structure. The invention also relates to a method for analysing biological samples and a test kit for sampling such material. The test kit typically contains a sampling device, which comprises a part of fibrous material, to which biological material may be collected or absorbed, and a pretreatment agent containing hydrolase activity, with which the sample material absorbed in the fibrous material may be released for analysis.

Description

Method for Analysing Samples from a Carbohydrate Matrix
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.
According to such a method 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. Thus, 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.
During contagious diseases pathogenic microbes proliferate, usually locally, in the body of a human being or an animal. For example, the bacterium that causes tonsillitis, a group A streptococcus, forms bacterial growth on the mucous membranes of the pharynx. These streptococci 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). Usually the sample bound to the cotton matrix of the sampling device is transferred to a liquid phase before analysis. 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 part of the material and by growing a culture from the matrix as such or by extracting some of the sample in the matrix into a liquid. According to known techniques cotton or viscose sticks and sampling pads are extracted with various acid and buffer solutions which may contain detergents, for example. This kind of vat liquor may impair the analyte to be determined from the sample, for example, by causing weaker growth of the microbes or by changing the structure of the antigens to be tested.
In order to obtain reliable results from the analysis, it is essential to take a sufficiently large and representative sample. In practice, a large fraction of the sample remains bound to the sampling device. If the concentration of the analyte in the sample is also low, the analyte may remain undetected. This is particularly adverse in the case of clinical samples where false negative results may cause an illness to be left untreated.
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. In connection with the invention it has surprisingly been observed that 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. Essentially more material to be examined is released from the carbohydrate matrix containing sample with the enzyme treatment than with treatment of the sample with acid or buffer solutions according to previously known techniques. Although the mechanism of this phenomenon is not exactly known, loosening of the matrix and consequent efficient release of the analyte may constitute one factor improving the reliability of the analyses. As is described in the examples presented below, this effect is not caused by the complete degradation or dissolution of the carbohydrate matrix because only small amounts of soluble sugars have been detectable. More specifically, the method according to the invention for pretreating a carbohydrate based matrix is characterized by what has been set forth and described in the characterizing part of Claim 1.
On the other hand, the method according to the invention for analysing biological material is characterized by what has been set forth and described in the characterizing part of Claim 20.
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.
As has been described herein above, 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. In the following, such a sample is also referred to with the term "carbohydrate- based (analyte) sample".
"Biological 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. Thus, the term 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.
Also 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. When the carbohydrate structure of the sample has first been modified enzymatically, the pathogen (virus, bacterium, fungus) may be more easily extracted and isolated from the sample to be analysed.
However, 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.
The term "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). Also 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.
A common feature of the carbohydrate-based matrices is that they contain bonds susceptible to hydrolysis, such as ester linkages and glycosidic linkages. The term "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. According to the present invention 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. Most suitably 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.
According to the method of the invention at least one preselected property (analyte) 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). Of these, 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. Thus, 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. Thus, it contains one or more of the following activities: 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. When the sample as itself comprises a carbohydrate-based matrix, 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.
5 mg - 50 mg of the enzyme product is used per a gram (of dry weight) of the sample. 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 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.
In a number of cases 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. In some cases in which the sample itself comprises a carbohydrate based matrix a sampling device is not needed, but the treatment is performed directly to the sample. 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.
According to a preferable particular embodiment 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. In this case 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
After the pretreatment according to the invention the sample is analysed in a way that as such is previously known in the art. Thus, 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. During the cultivation stage the sample may be cultivated by diluting onto a dish, whereby single bacteπa yield separate colonies which may be identified Also 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. However, in principle any microbe or virus that can be cultivated or otherwise identified is suitable for determination by to the method according to the invention.
The invention has several advantages. Thus, 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.
In the following, the invention is described in detail with reference to the enclosed figures and some application examples.
In figure 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.
Example 1
Characterisation of the enzymes
Some enzyme products suitable for use in the method according to the invention are described in this example.
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). There are several commercial products available which have been produced, e.g., by fungi of the strains Trichoderma or Humicola. Here a preparation suitable for use for the purpose described above and with the tradename Econase (Primalco Ltd.) is described in more detail. The manufacturer gives the following activity information for the product: FPU (filter paper unit) 31 U/ml
HEC (hydroxyethyl cellulose) 13 500 nkat/ml β-glucosidase 1 030 nkat/ml
In figure 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).
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 Additionally, also 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
There are several commercially available enzyme preparations which contain enzymes that break down or modify the pectin matter in the cell walls of plants In this connection a commercial product called Pectinex Ultra was used, containing at least the following activities
pectin esterase E C 3 1 1 1 1 polygalacturonase E C 3 2 1 15 exopolygalacturonase E C 3 2 1 67 pectin lyase E C 4 2 2 2 endoglucanase E C 3 2 1 4 endoxylanase E C 3 2 1 8 mannanase E C 3 2 I 78
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
Example 2
Dispersion of an Omni-SAL sampling device with cellulase
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.
In figure 2 the breakdown of the structure of the sampling pad
Figure imgf000013_0001
is shown as a function of time with different enzyme dilutions, and with the buffer and the Omni-SAL solution.
It can be seen in Figure 2 that the structure of the sampling pad disintegrates significantly with all enzyme dilutions, but practically not at all with just buffer or Omni-SAL solution.
When the sample solution (enzyme dilution 1: 1,000) was analysed for soluble sugars with liquid chromatography after 6 hours of incubation, the surprising observation was made that only about 10 % of the cellulose had disintegrated into glucose due to the action of 12 the enzymes. Thus the breakdown of the structure of the sampling device or dispersion of the pad took place due to the action of the enzymes before the actual hydrolysis of cellulose. For example, after one (1) hour, which for practical reasons is a suitable incubation time, the amount of soluble sugars is minute, but the opening of the cellulose structure can be observed clearly.
Example 3
The effect of enzyme treatment on the cultivation result of a biofilm sample collected with a cotton stick
The growth of biofilms took place on stainless steel plates (AISI 304/2B). The steel plates were washed with warm (50 °C) 2 % RBS 35 detergent (Chemical Products SPRL, Belgium), rinsed five times with warm water and sterilized in an autoclave. The following test organisms were grown on the steel plates:
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 108-109 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 (three cotton sticks per treatment) 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.
The results are shown in Table 1 :
Table 1. Effect of enzyme treatment on the growth of a biofilm sample.
Organism Detached colonies, log cfu/cm2, after sample treatment with cellulase pectinase mixture buffer of pectinase and cellulase
P.fragi 5 49+0.32 5.45+0 44 5 33+0.47 4.09+0 44
B. lichemformis 4 44±0.32 4 73+0 41 4.94+0.42 4.35+0.29
P. inopmatus 3 46+0.42 3 04+0 15 2.98+0 31 2.72+0 24
C. utilis 3.97+0 44 3 61+0 29 3 74+0.29 3.69+0.37
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
Example 4
The effect of enzyme treatment on the function of the Strep A instant test
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 μl of a Streptococcus pyogenes strain grown over night at a concentration of 106 or 105 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
The results are presented in Table 2
Table 2. The effect of enzyme treatment on the function of a Strep A instant test.
Figure imgf000017_0001
1 +++, blue, ++, light blue, +, faint blue, -, no colour
2 NT, not tested
The conclusion is that when using a cotton stick as the sampling device the enzyme treatment enhances the release of bacteria from the sampling device, which increases the sensitivity of the antigen determination test At a lower concentration of bacteria the enzyme treated cotton stick gave a better result than the dacron stick used in the commercial test
Example 5
The effect of enzyme treatment on the determination of the Chlamydia antigen
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 antigen solution was absorbed into a sample stick, whereafter the stick was placed in a sterile tube and was left to stand there overnight at room temperature (= transport of the sample to the laboratory) For the pretreatment of the cotton stick the enzyme solution (cellulase) was diluted (1/100 and 1/500) into dilute HC1 with at pH 5.0. An enzyme buffer (dilute HC1) without enzyme was used as the negative control. The cotton sticks were shaken in the enzyme solution in test tubes with a shaker at room temperature for 1 hour, whereafter the enzyme solutions were analysed according to the instructions of the Wellcozyme Chlamydia kit for EIA assay. Two sticks were subjected to each treatment, and two replicate EIA determinations were made from each stick. The results were compared to the EIA results obtained without pretreatment (the cotton stick was extracted directly according to the instructions of the kit).
The results are presented in Table 3.
Table 3. The effect of cellulase treatment on the determination of the Chlamydia antigen
Pretreatment EIA absorbance x 1000
Cellulase 1/100 1,694
Cellulase 1/500 2,512
Enzyme buffer 1,078
No treatment 1,779
The cellulase treatment (1:500) clearly increased the sensitivity of the test.
Example 6
The effect of enzyme treatment on the dispersion of excrement
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.
Table 4. The effect of enzyme treatment on the dispersion of excrement.
Enzyme Absorbance (A^^)
30 min. 60 min 120 min. 480 min.
Cellulase 0.020 0.060 0.120 0.260
Hemicellulase 0.040 0.110 0.220 0.345
Cellulase 0.010 0.040 0.080 0.160
+ hemicellulase
Additionally, the enzyme treated samples were plated on agar nutrient and incubated for 2 days at 36 °C. This enabled the determination of the total numbers of endogenic, aerobically growing bacteria in the samples. The results are reported in Table 5.
18
Table 5. The effect of enzyme treatment on the number of bacteria grown from excrement
Treatment Concentration of bacteria (x 105) 60 min. 480 min.
No enzyme 0.50 0.250
Cellulase 1.25 1.60
Hemicellulase 1.25 1.60 Mixture of cellulase and hemicellulase 3.00 3.20
As can be seen from Table 4, all the enzyme treatments significantly enhanced the dispersion of excrement. The dispersion of excrement caused by the enzymes also clearly increased the number of bacteria that could be cultivated from the excrement (Table 5).

Claims

Claims
1. A method for pretreating a carbohydrate-containing analysis sample, characterized in that - the sample is brought into contact with an enzyme preparation containing cellulase, hemicellulase, amylase and/or pectinase activity, which preparation affects the structure of the carbohydrate matrix.
2. A method according to Claim 1, characterized in that the sample is brought into contact with such an amount of enzyme that is sufficient to loosen the matrix structure.
3. A method according to Claim 1 or Claim 2, characterized in that the sample is brought into contact with an amount of enzyme that does not essentially dissolve the matrix.
4. A method according to Claim 3 for treating a sample containing cellulosic fibres, characterized in that at the most about one fifth of the cellulosic fibres disintegrate to mono- or oligosaccharides.
5. A method according to Claim 1, characterized in that an enzyme preparation with cellobiohydrolase and endoglucanase activity is used.
6. A method according to Claim 1, characterized in that an enzyme preparation containing xylanase and/or mannanase activity is used.
7. A method according to Claim 5 or Claim 6, characterized in that an enzyme preparation containing cellulase and hemicellulase activity, especially cellulase and xylanase and/or mannanase activity, is used.
8. A method according to Claim 1, characterized in that a pectinase preparation affecting the galacturonic acid, rhamnose, xylose, fructose, arabinose, galactose, xyloglucan and/or arabinoglucan components of the cell wall of the plant fibre, is used.
9. A method according to Claim 8, characterized in that a pectinase preparation containing one or more of the following activities: pectin esterase, polygalacturonase, exopolygalacturonase, pectin lyase, endoglucanase, endoxylanase and mannanase.
10. A method according to any of the preceding Claims, characterized in that 5 mg - 50 mg of the enzyme preparation is used per a gram of (dry weight of) sample.
1 1. A method according to any of the preceding Claims, characterized in that the sample is submerged into or mixed with the enzyme solution.
12. A method according to any of the preceding Claims, characterized in that the enzyme treatment is performed at 0 - 60 °C for 1 minute - 48 hours and at pH 3 - 10, preferably at about 3 - 8.
13. A method according to any of the preceding Claims in which at least one preselected property is determined from the sample after the pretreatment, characterized in that microorganisms are determined from the sample.
14. A method according to Claim 13, characterized in that bacteria, fungi, yeasts, protozoa and/or viruses contained in the sample are determined.
15. A method according to Claim 13 or Claim 14, characterized in that a microorganism or rota- or adenovirus, belonging to the species Pseudomonas, Pediococcus, Bacillus, Candida, Streptococcus, Staphylococcus, Escherichia, Salmonella, Campylobacter or Chlamydia, is determined from the sample.
16. A method according to any of the preceding Claims 13 - 15, characterized in that microorganisms are determined by growing a culture or by analysing their antigens.
17. A method according to any of the preceding Claims 13 - 15, characterized in that microorganisms are determined by antibodies that react with them. PC17FI97/00368
21
18. A method according to any of the preceding Claims 1 - 12, in which at least one preselected property is determined from the sample after the pretreatment, characterized in that hormones, markers, prions, trace elements and/or antibodies are determined from the sample.
19. A method for analysing biological material, according to which method
- a sample is taken from the material, and a preselected property is determined from the sample, characterized in that - the sample is taken with a sampling device consisting of plant fibres, and
- the sampling device is treated after sampling, before determining the selected property, with an enzyme product containing hydrolase activity.
20. A method according to Claim 19, characterized in that the sampling unit is a cellulosic matrix which has possibly been attached to a holder suitable for sampling.
21. A method according to Claim 20, characterized in that the cellulosic matrix is cotton, viscose or cellulosic mass or a product prepared by chemically modifying these.
22. A method for analysing biological material containing a carbohydrate-based matrix, according to which method
- a sample is taken from the material, from which sample a preselected property is determined, characterized in that - the sample is treated after sampling and before determining the selected property with an enzyme preparation containing hydrolase activity.
23. A method according to Claim 22, characterized in that the sample comprises excrement.
24. A method according to Claim 22, characterized in that the sample comprises bacterial plaque or mucus contained in sputa or pharynx, saliva or cervical samples. 22
25. A method according to Claim 22, characterized in that the sample comprises plant material.
26. A test kit for taking samples from biological material, which kit contains - a sampling device, which comprises a part of fibrous material, to which biological material may be collected or absorbed, and
- an agent for pretreatment, with which sample material absorbed in the fibrous material may be released for analysis, characterized in that - the pretreatment agent comprises an enzyme product with hydrolase activity.
27. A test kit according to Claim 26, characterized in that it also contains analytical equipment for analysing the sample material.
28. A test kit according to Claim 26 or Claim 27, characterized in that the pretreatment agent contains an enzyme preparation that affects a carbohydrate matrix, which preparation has hydrolase activity and a buffer stabilizing the action of the enzymes and a protecting agent.
29. The use of an enzyme preparation containing cellulase, hemicellulase, amylase and/or pectinase activity, which product affects the structure of a carbohydrate matrix, for the pretreatment of analysis samples.
PCT/FI1997/000368 1996-06-11 1997-06-11 Method for analysing samples from a carbohydrate matrix WO1997047764A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI962408 1996-06-11
FI962408A FI101809B (en) 1996-06-11 1996-06-11 Ways of analyzing a sample from a carbohydrate matrix

Publications (1)

Publication Number Publication Date
WO1997047764A1 true WO1997047764A1 (en) 1997-12-18

Family

ID=8546184

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI1997/000368 WO1997047764A1 (en) 1996-06-11 1997-06-11 Method for analysing samples from a carbohydrate matrix

Country Status (2)

Country Link
FI (1) FI101809B (en)
WO (1) WO1997047764A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079527A2 (en) * 2000-04-18 2001-10-25 Pearl Technology Holdings, Llc Sexual fidelity and sex crime verification
WO2001090297A1 (en) * 2000-05-25 2001-11-29 Cellomeda Oy Device intended for sample collection in diagnostics of diseases and its use
WO2010043668A1 (en) * 2008-10-17 2010-04-22 Zentech S.A. Dried blood spots for blood analysis
WO2016071805A1 (en) * 2014-11-07 2016-05-12 Stora Enso Oyj Improved method for determination of microorganisms
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 (en) * 1984-02-27 1985-09-04 Becton Dickinson and Company Diagnostic test for streptococcus 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 (en) * 1984-02-27 1985-09-04 Becton Dickinson and Company Diagnostic test for streptococcus 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 (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079527A2 (en) * 2000-04-18 2001-10-25 Pearl Technology Holdings, Llc Sexual fidelity and sex crime verification
WO2001079527A3 (en) * 2000-04-18 2002-08-15 Pearl Technology Holdings Llc Sexual fidelity and sex crime verification
WO2001090297A1 (en) * 2000-05-25 2001-11-29 Cellomeda Oy Device intended for sample collection in diagnostics of diseases and its use
WO2010043668A1 (en) * 2008-10-17 2010-04-22 Zentech S.A. Dried blood spots for blood analysis
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 (en) * 2014-11-07 2016-05-12 Stora Enso Oyj Improved method for determination of microorganisms
CN107027314A (en) * 2014-11-07 2017-08-08 斯道拉恩索公司 Determine the improved method of microorganism
JP2017532977A (en) * 2014-11-07 2017-11-09 ストラ エンソ オーワイジェイ Improved method for the determination of microorganisms
EP3215632A4 (en) * 2014-11-07 2018-06-20 Stora Enso Oyj Improved method for determination of microorganisms
US10017806B2 (en) 2014-11-07 2018-07-10 Stora Enso Oyj Method for determination of microorganisms
CN107027314B (en) * 2014-11-07 2021-02-19 斯道拉恩索公司 Improved method for determining microorganisms

Also Published As

Publication number Publication date
FI101809B1 (en) 1998-08-31
FI962408A (en) 1997-12-12
FI962408A0 (en) 1996-06-11
FI101809B (en) 1998-08-31

Similar Documents

Publication Publication Date Title
US5627045A (en) Multi-test format with gel-forming matrix for characterization of microorganisms
Sandrock et al. Fungal sensitivity to and enzymatic degradation of the phytoanticipin α-tomatine
Cody Distribution of chitinase and chitobiase in Bacillus
BUTLER et al. Isolation conditions for high yields of protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae)
CA2557487C (en) Detection of contaminants retained on a filter by a detectable moiety-producing substrate
CA2500464C (en) Method for detecting and counting micro-organisms in a sample
Pegg et al. Chitinase activity in Lycopersicon esculentum and its relationship to the in vivo lysis of Verticillium albo-atrum mycelium
Samriti et al. Garbage enzyme: A study on compositional analysis of kitchen waste ferments
Hagerman et al. Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores
US5882882A (en) Gel matrix with redox purple for testing and characterizing microorganisms
WO1997047764A1 (en) Method for analysing samples from a carbohydrate matrix
Singh et al. Evaluation of biomass
Safo-Sampah et al. Polysaccharide-hydrolyzing enzymes of Frankia (Actinomycetales)
JP4528939B2 (en) Biosensor storing soil microorganisms and use thereof
Bauchop et al. Gut microbiology and carbohydrate digestion in the larva of Costelytva zealandica (Coleoptera: Scarabaeidae)
CA2279378C (en) Method of selectively determining a fungal biomass
Nagy et al. Optimization of conditions for culture of the test bacteria used for direct bioautographic detection 1. The gram-positive test bacterium Bacillus subtilis
Rezaeian et al. Methods for the isolation, culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems
Fay et al. A scanning electron microscopy study of the invasion of leaflets of a bloat-safe and a bloat-causing legume by rumen microorganisms
Cavalitto et al. Quantification of protopectinase SE, an endopolygalacturonase with pectin-releasing activity from Geotrichum klebahnii
JPH11513901A (en) Methods and kits for pretreatment of a target surface
Takeda et al. Cell-wall chemistry, structure and components
Lisker et al. Effect of propagule size on the in vitro production of polygalacturonase and cellulase by Rhizoctonia solani
KR920008386B1 (en) Detection paper of staphylococcus aureus
Beattie Incidence and importance of Bacillus species in raw milk and in the dairy environment

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 98501255

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase