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WO1991019003A1 - Procede de detection - Google Patents

Procede de detection Download PDF

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Publication number
WO1991019003A1
WO1991019003A1 PCT/AU1991/000247 AU9100247W WO9119003A1 WO 1991019003 A1 WO1991019003 A1 WO 1991019003A1 AU 9100247 W AU9100247 W AU 9100247W WO 9119003 A1 WO9119003 A1 WO 9119003A1
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WO
WIPO (PCT)
Prior art keywords
contaminant
cell
antibodies
sample
process according
Prior art date
Application number
PCT/AU1991/000247
Other languages
English (en)
Inventor
Lester John Farr
Vincent Habib Atrache
Geoffrey Harold Braid
David Ernest Forester Harrison
Original Assignee
Biotech Australia Pty. Limited
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 Biotech Australia Pty. Limited filed Critical Biotech Australia Pty. Limited
Publication of WO1991019003A1 publication Critical patent/WO1991019003A1/fr

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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

Definitions

  • the present invention relates to processes for the detection of contaminants in samples, such as bacteria, yeasts or fungi, in, for example, food, beverage, water, dairy, cosmetic or pharmaceutical samples.
  • Methods for the detection of microorganisms in samples include use of devices which detect the metabolic activities of microorganisms by electrical, chemical, or biochemical means, for example those which use a cell connected to an electrical circuit with a means for the measurement of variations in impedance or conductivity in the cell to give an indication of growth within the cell.
  • Examples of electrical detection devices for use in these methods include devices such as the Bactometer TM
  • indirect detection systems include those which detect changes in oxygen concentration or production of gaseous or soluble metabolites or pH changes or redox or enzymatic reactions.
  • Electrical detection devices for the direct enumeration of microorganisms based on the electrical resistance produced by non-conductive particles suspended in an electrolyte are also known.
  • An example of such a device is the Coulter counter. At present such instruments cannot be used for the detection of specific microorganisms present in a mixed population.
  • cell is used herein to describe a receptacle which may include a lid and may include baffle(s) depending into the receptacle from the lid or from interior surface(s) of the receptacle.
  • the present invention provides rapid and accurate detection processes with the benefits of immunoenrichment and with the low operator involvement and automation of detection systems such as the Bactometer TM or Coulter count .erTM
  • a process for the detection and/or quantification of at least one contaminant having metabolic activity, in a sample comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant; inoculating the cell with the sample; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.
  • the process for the detection and/or quantification of at least one contaminant having metabolic activity, in a sample comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant and are capable of binding the at least one contaminant without adversely affecting its ability to replicate; inoculating the cell with the sample for a sufficient time to allow binding of the at least one contaminant to the antibodies; removing unbound sample; introducing a medium for the growth of the at least one contaminant into the cell; incubating the cell under conditions suitable for the growth of the at least one contaminant; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.
  • the unbound sample is removed by washing.
  • the process additionally comprises incubating the sample in a suitable medium to resuscitate the at least one contaminant prior to inoculating the sample into the cell.
  • the process additionally comprises a preenrichment step which comprises: exposing the sample to a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant, which antibodies are specific for the at least one contaminant, to allow binding of the at least one contaminant to the antibodies; removing unbound sample from the substrate ; followed by releasing the bound at least one contaminant from the substrate with a. releasing agent; and introducing the released at least one contaminant into the cell, for the detection process.
  • a preenrichment step which comprises: exposing the sample to a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant, which antibodies are specific for the at least one contaminant, to allow binding of the at least one contaminant to the antibodies; removing unbound sample from the substrate ; followed by releasing the bound at least one contaminant from the substrate with a. releasing agent; and introducing the released at least one contaminant into the cell, for the detection process.
  • the preenrichment step may comprise incubating the sample in a medium specific for the growth of the at least one contaminant to specifically increase the numbers of the at least one contaminant with respect to other contaminants which may be present in the sample, prior to inoculating the sample into the cell.
  • the process of the first embodiment may alternatively comprise: providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant 5 which antibodies are specific for the at least one contaminant; exposing the sample to the solid substrate for a
  • the substrate is washed to remove unbound sample prior to introducing the substrate into the cell.
  • the process comprises: 20 introducing a medium for the growth of the at least one contaminant; and incubating the cell under conditions suitable for the growth of the at least one contaminant on introducing the substrate into the cell, and the antibodies are capable of 25 binding the at least one contaminant without adversely affecting its ability to replicate.
  • resuscitation and/or preenrichment steps could also be included, prior to 30 exposure of the sample to the solid substrate.
  • the metabolic activity may be detected by a variation in impedance or conductivity in the cell, a change in oxygen concentration or the production of gaseous soluble 35 metabolites, or changes in hydrogen ion concentration or changes in redox potential or enzymatic activity or other means of measuring metabolic activity.
  • a variation in impedance or conductivity the presence of the at least one contaminant could be detected by measurement of the polarization which occurs as the at least one contaminant is bound by the antibodies.
  • a process for the direct detection and/or enumeration of at least one contaminant in a sample comprises: providing a cell having a surface to which the sample is exposed carrying antibodies specific to the at least one contaminant; inoculating the cell with the sample for an appropriate period to allow binding of the at least one contaminant to the antibodies; removing unbound material from the cell; adding an electrolyte solution to the cell; adding a releasing agent to release the bound at least one contaminant; and detecting the at least one contaminant by measuring the change in electrical resistance of the electrolyte solution on release of the bound at least one contaminant from the cell surface.
  • the process comprises washing the cell to remove unbound material from the cell.
  • the process comprises: adding a growth medium for the at least one contaminant to the cell; incubating the cell for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; and removing the growth medium prior to adding the electrolyte solution to the cell and the antibodies are capable of binding the at least one contaminant without adversely affecting its ability to replicate.
  • the growth medium is removed by washing.
  • preenrichment and resuscitation steps could be incorporated, prior to inoculating the sample into the cell.
  • the process of the second embodiment may alternatively comprise: providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant; exposing the sample to the solid substrate for a sufficient time to allow binding of the at least one contaminant to the antibodies, or binding to the antibodies and growth; inserting the substrate into a cell; adding an electrolyte solution to the cell; adding a releasing agent to the cell to release the bound at least one contaminant; and detecting the at least one contaminant by measuring the change in electrical resistance of the electrolyte solution in the cell on release of the bound at least one contaminant from the substrate.
  • the process may additionally comprise introducing the substrate with bound at least one contaminant into a cell containing a growth medium for the at least one contaminant for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; and removing the growth medium prior to exposure to the electrolyte solution, and in this form the antibodies should be capable of binding the at least one contaminant without adversely affecting its ability to replicate.
  • pre-enrichment and/or resuscitation steps may be used prior to exposure of the sample to the solid substrate.
  • Electrolyte solutions suitable for use in the processes of the second embodiment include physiological saline and phosphate buffered saline.
  • the sample to be analysed in a process of the invention is a food, beverage, water, dairy, cosmetic or pharmaceutical sample, which may be prepared for analysis by standard methods of sample preparation.
  • the at least one contaminant to be detected in a process of the invention is a microorganism, such as a bacterium, yeast or fungus.
  • Other contaminants include other prokaryotic and eukaryotic single celled organisms.
  • the contaminant is a bacterium selected from Salmonella species and Listeria species, particularly Salmonella typhimurium or Listeria monocvto ⁇ enes.
  • growth media are used it is preferred that they are selective media.
  • a selective growth medium may enhance the specificity of the process.
  • the antibody is raised against a surface antigen of the microorganism. More preferably, the surface antigen is a flagellar protein or lipopolysaccharide.
  • the removal step is preferably one which includes washing.
  • the invention provides for processes wherein the antibody is coated onto the interior walls of the cell, and those where the cell is provided with a separate antibody support such as a cap having dependent baffles coated with 5 antibody.
  • a volume of the pre-enrichment broth is removed and placed in a receptacle, which may be purpose 15 made.
  • a receptacle which may be purpose 15 made.
  • the "ImmunoCap” which seals the receptacle and captures specific microorganism species using specific antibodies immobilised on the suspended support system.
  • the "ImmunoCap” is then transferred to the well of an electrical detection device, eg. a Bactometer TM or Coulter counter TM. This is then inserted into the standard incubation system for the detection device and variations in iimmppeeddaannccee oorr ccoonndduuccttiivviittyy ccaauusseedd bbyy ggeenneerraattiioonn ooff cchhaarreged 5 metabolites are measured in the case of the Bactometer .r
  • the removable support can be taken from the assembly. This can then be used to perform an ELISA type reaction or other form of secondary analysis to 35 confirm or otherwise the presence or absence of the specific organism or organisms. Secondary analysis may be used at the end of any version of the process.
  • the sample is added to a cell coated with antibody and incubated so that microcolonies of bound microorganism form. After washing to remove unbound material the antibody-bound microcolonies are detected by measuring variations in impedance or conductivity in the cell caused by generation of charged metabolites or by changes in electrical resistance caused by release of the microcolonies from the cell surface.
  • the microcolonies may alternatively be detected directly.
  • the sample to be analysed may be subjected to preenrichment and/or resuscitation steps.
  • Some versions of the processes of the invention contemplate the release of bound contaminants.
  • Contaminants bound to the antibodies may be released from the antibodies, or the antibody-contaminant complex may be liberated from the cell surface.
  • Releasing agents suitable for dissociating the antibody-contaminant bond include:
  • chaotropic agents such as 4.5 M MgCl 2 pH 7.5 or 2.5M Nal pH 7.5
  • polarity reducing agents such as ethylene glycol in solutions of up to 50%
  • pH change inducing agents such as glycine/HCl pH 2.5, aqueous H 3 pH 11 or 0.5% KOH pH 12.5.
  • the releasing agent is 0.5% KOH pH12.5.
  • Releasing agents suitable for dissociating the antibody-contaminant complex from the cell or substrate surface include papain and similar proteolytic enzymes.
  • the released contaminant can be measured directly by Coulter Counter. Release is also desirable in those processes which include a step whereby the released contaminants are plated out for colony counts or are subjected to other analysis such as ELISA or fluorescent immunoassay. Contaminants may be released from the
  • ImmunoCap baffle or dipstick for plating if desired or for adding to an ELISA well, or other secondary analysis, as well as for direct detection.
  • FIGURE 1 A first figure.
  • Figure la shows a single ImmunoCap to fit a Bactometer type of well.
  • Figure lb shows a bank of ImmunoCaps.
  • Figure lc shows a well designed to hold 5 to 10 mL of enrichment broth.
  • Figure Id shows a bank of wells, each designed to hold 5 to 10 mL of enrichment broth.
  • FIG. le shows a further enhancement whereby a removable baffle can be an integral part of the ImmunoCap.
  • This baffle can be coated with antibodies to a specific species/strain which can be removed for confirmation.
  • FIG. 1 shows a further enhancement of the ImmunoCap shown in Figure le with a different antibody coated baffle configuration.
  • FI G URE i shows an inverted plan (undersi d e ) view of the ImmunoCap as shown in Figure if.
  • FI G URE 2a shows a schematic representation of a process of the invention using an ImmunoCap as shown in Figure la.
  • the process involves a first step of resuscitation/pre-enrichment. This is followed by the specific immunocapture of a putative contaminant using the Immuno C ap of Figure la.
  • the final step in the process involves placing the ImmunoCap into the well of an electrical detection device and measuring changes in impedance ' or conductivity.
  • FI GU RE 2b shows a typical positive result for the process of Figure 2a.
  • FI GU RE 2c shows a typical negative result for the process of Figure 2a.
  • FI G URE 4a this shows a schematic representation of another process of the present invention, wherein the a b sence or presence of micro-colonies are detected by changes in impedance or conductivity.
  • FIGURE 4b shows a typical positive result for the process of Figure 4a.
  • FIGURE 4c Shows a typical negative result for the process of Figure 4a.
  • Example 3 Shows a graph representation of the results of Example 3 at 10 Salmonella per ml.
  • the graph shows typical conductance curves demonstrating comparative detection time for Salmonella following immunoenrichment using antibody coated dipstick (— ) and casein control coated dipstick fixed.
  • Example 3 Shows a graph representation of the results of 7 Example 3 at 10 Salmonella per ml.
  • the graph shows typical conductance curves demonstrating comparative detection time for Salmonella following immunoenrichment using antibody coated dipstick ( ) and casein control coated dipstick fixed.
  • Standard devices are available for the automated detection of microbial contaminants in samples.
  • the processes of the first embodiment of the present invention can use automated detection devices such as the Bactometer , the Malthus or the Rabit, or pH detection units, or units for the detection of oxygen depletion, or for detection of production of gaseous or soluble
  • the antibodies for use in the processes of the invention can be either obtained commercially or prepared using standard techniques for the raising of antibodies.
  • the use of polyclonal and monoclonal antibodies is included.
  • mixtures of antibodies may be desirable, for instance, where different species or serotypes are to be detected, but not distinguished between in the sample being tested.
  • Antibodies can be tested for their specificity and/or their effect on the replication of bound contaminants by standard techniques.
  • the binding of the antibodies to the cell surfaces or substrate surfaces can be performed by standard techniques.
  • Resuscitation, pre-enrichment and growth of the samples can be performed by standard techniques of microbiology for the processes of the invention.
  • the samples or cells or baffles may be exposed to subsequent detection procedures such as ELISA techniques, or plating techniques.
  • Alternative methods which may be used include immunofluorescent techniques using whole fluorescent labelled antibodies or fluorescent labelled Fab fragments, or colorimetric detection processes.
  • Each of the secondary analysis techniques can be performed using methods and reagents standard in the appropriate arts.
  • Secondary detection methods can be used for confirming the presence and/or quantity of the contaminants identified, in any of the processes of the invention.
  • Release of contaminants or contaminant-antibody complexes from cells, substrates or baffles can be performed using standard techniques employing suitable dissociating agents.
  • Agents suitable for dissociating the antibody- contaminant bond include: heat; chaotropic agents such as 4.5M MgCl_ pH 7.5 or 2.5M Nal pH 7.5; polarity reducing agents such as ethylene glycol in solutions of up to 50%; and pH change inducing agents such as glycine/HCl pH 2.5, aqueous H 3 pH 11 or 0.5% KOH pH 12.5.
  • chaotropic agents such as 4.5M MgCl_ pH 7.5 or 2.5M Nal pH 7.5
  • polarity reducing agents such as ethylene glycol in solutions of up to 50%
  • pH change inducing agents such as glycine/HCl pH 2.5, aqueous H 3 pH 11 or 0.5% KOH pH 12.5.
  • Agents suitable for dissociating the antibody-contaminant complex from the cell or substrate surface include papain and similar proteolytic enzymes.
  • the process of the second embodiment of the present invention can use automated detection devices such as the Coulter Counter. These processes involve the direct . detection of the contaminant rather than detecting a product or an effect generated by the contaminant. - 15 - Since the initial capture is carried out using specific antibodies for the contaminant of interest then any change in electrical measurement will be due to that contaminant. In other words, the risk of another type of contaminant giving an interfering response is virtually eliminated, and hence the false positive rate is substantially reduced.
  • incubation conditions selection of growth media and electrolytes can all be performed by standard techniques.
  • the choice of incubation conditions and growth media will, of course, be influenced by the nature of the contaminant(s) being detected.
  • Washing steps are performed with standard washing buffers compatible with the contaminants and antibodies.
  • the choice of suitable releasing agents can be performed by standard techniques. This choice may be influenced by whether it is desirable to keep the contaminant alive for secondary analysis.
  • the cells for use in the processes of the invention may comprise commercially available well units such as those provided for use with a Bactometer or Coulter Counter.
  • the cells may be purpose made from materials suitable for binding antibodies and holding samples for analysis.
  • substrates for carrying antibodies could comprise commercially available dipsticks either with or without preformed antibody coatings or baffles or paddles made from suitable materials for antibody coating.
  • Materials suitable for use include polystyrene, polyvinyl chloride, nylon, nitrocellulose, agarose and titanous hydroxide. - 16 -
  • Figure 2 shows a process of the invention in schematic form.
  • the contaminant in this case is a bacterium.
  • the sample to be tested is subjected to a resuscitation/pre-enrichment step which allows damaged cells to become revived prior to analysis.
  • The-- pre-enrichment step improves the sensitivity of the assay, concentrating the organism of interest in the sample.
  • the next step incorporates the use of the ImmunoCap to selectively capture organisms of interest using the specific antibodies on the baffles. This takes place by transferring 5 to 10 mL of the pre-enrichment broth into the ImmunoCap well and incubating for a short period of time.
  • the ImmunoCap is now removed and washed if necessary, before placing in the Bactometer or equivalent well containing electrodes and a suitable broth medium.
  • the wells are next placed in the incubator unit and the impedance of the broth measured as normally carried out in the Bactometer. During this time, microcolonies will grow on the surface of the baffles and their metabolites will cause a change in the impedance or conductance of the broth medium.
  • Figure 3 shows how a presumptive positive result from the Bactometer can be confirmed using the ImmunoCap with removable baffle (Figure le,lf).
  • the removable baffle coated with specific antibodies to a particular species of organism eg. Listeria monocytogenes or Salmonella typhimurium is placed into a tube (or the Bactometer well could be reused if washed).
  • a particular species of organism eg. Listeria monocytogenes or Salmonella typhimurium
  • the baffle has specifically captured this species and microcolonies grow.
  • the baffle is now - 17 - washed, incubated with a specific antibody-conjugate, washed and further incubated with a substrate.
  • Generation of a colour on the baffle acts as confirmation of the presence of the specific species of microorganism etc.
  • the bound cells can be released from the baffle and added to an ELISA well if the Optical Density (OD) is required to be measured, or released onto an agar plate etc if formation of colonies is required.
  • OD Optical Density
  • FIG 4 shows a process of the invention in schematic form.
  • the contaminant is a microorganism such as a bacterium.
  • the sample to be tested is subjected to a resuscitation step or a preenrichment step whereby a baffle carrying antibodies specific for the microorganism of interest is inserted into the sample to bind the microorganism of interest.
  • the baffle may be washed and then the microorganism is released into the cell which in this case is a Bactometer well carrying antibodies, specific to the microorganism, which do not adversely affect the ability of the microorganism to replicate, on the interior side walls of the well.
  • the cell is incubated to allow binding of the microorganism to the antibodies and growth, the cell containing a medium which may be selective or non-selective for the growth of the microorganism.
  • the Bactometer unit is used to monitor growth. As the sample is added the conductivity rises and plateaus as microorganisms are bound by antibodies. The broth is removed, the cell washed and fresh medium added. The conductivity declines at first but as growth of bound organisms occurs, the conductivity increases. - 18 - If the contaminant is not present the increase in conductivity after washing does not occur.
  • a Bactometer impedance/conductance detection system (Bactometer M128 - Vitek Systems 595, Anglum Drive, Hazelwood, Missouri, USA) was evaluated for the automated detection of Salmonella, following initial capture on ImmunoCap dipsticks.
  • Nunc Maxisorp Immunosticks (A/S Nunc-Postbox 280-Kamstrup-DK4000 Roskilde-Denmark.) were incubated in a solution containing 30 ⁇ g/mL sheep anti-Salmonella flagella antibodies in lOmM Phosphate buffer pH8.1, for 24 hours at room temperature ( 22°C) . The sticks were then washed once with water and then incubated in a solution containing 0.1% gelatine in lO ⁇ m phosphate buffer, pH8.1 overnight at room temperature to block any remaining sites on the stick surface. The coated and blocked immunosticks were washed once with water and dried. The dried antibody coated sticks were used directly in the experiments described below. Anti-Salmonella flagella antibody coated dipsticks are also commercially available through Bioenterprises Pty Ltd., 28 Barcoo Street, Roseville NSW 2069, Australia.
  • Nunc Maxisorp Immunosticks were incubated in a solution of 1.75% casein pH7.0 for 24 hours at room temperature. The coated sticks were washed once with water then dried. - 19 - Salmonella tvphimurium (ATCC7823) and E L. coli (ATCC27325) were grown overnight at 37C, in buffered peptone water.
  • Dipsticks were added to each of the dilutions for 20 min at room temperature ( 22C) .
  • dipsticks were then washed 3 times with 4.5mL of buffered peptone water, before being fixed in position in the Bactometer wells, one to each well.
  • Each dipstick was immersed in a modified lysine decarboxylase media (Vitek Systems) and incubated at 37C during which time, continual conductance measurements were taken and the data captured.
  • Standard sofware version R02.1 (Vitek Systems 595, Anglum Drive, Hazelwood, Missouri, USA) was used to determine the detection times for the various concentrations of Salmonella on both control and antibody coated dipsticks.
  • Nunc Maxisorp Immunosticks (A/S Nunc-Postbox 280-Kamstrup-DK4000, Roskilde-Denmark.) were incubated in a solution containing 30 ⁇ g/mL sheep anti-Salmonella flagella antibodies in lOmM Phosphate buffer pH8.1, for 24 hours at room temperature ( 22°C). The sticks were then washed once with water and then incubated in a solution containing 1.75% casein in pH7.0 overnight at room temperature to block any remaining sites on the stick surface. The coated and blocked immunosticks were washed once with water and dried. The dried antibody coated sticks were used directly in the experiments described below. Anti-Salmonella flagella antibody coated dipsticks are also commercially available through Bioenterprises Pty Ltd., 28 Barcoo Street, Roseville NSW 2069, Australia.
  • Nunc Maxisorp Immunosticks were incubated in a solution of 1.75% casein pH7.0 for 24 hours at room temperature. The coated sticks were washed once with water then dried. - 21 - Salmonella tvphimurium (ATCC7823) and E-- COli (ATCC27325) were incubated overnight at 37°C, in Buffered Peptone Water.
  • Antibody-coated and casein control dipsticks were added in triplicate to each of the following dilutions and incubated for 20 min. at room temperature ( 22°C).
  • dipsticks were then removed and placed into tubes containing M-broth (DIFCO) (a non-specific growth medium) and incubated for 4 hours at 37°C.
  • DIFCO M-broth
  • the dipsticks were removed from the growth medium then washed to displace any non-specifically bound organisms.
  • the washed dipsticks were placed in an electrolyte solution and estimation of bacterial numbers in solution, before and after release of the organisms from the dipstick surface, made using the
  • the Salmonella bacteria were released from the dipstick surface into the electrolyte solution either by heating the dipstick in electrolyte at 100°C for 10 minutes or by treatment of the dipstick with papain lO ⁇ g/mL in 0.5M Acetate buffer pH5.2.
  • Aperture of 30 ⁇ m diameter was used, at 0.88-2.00 ⁇ m window using Isoton II electolyte as the diluent.
  • the present invention is of use in the field of quality control in the food, beverage, water supply, dairy, cosmetic and pharmaceutical industries.

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Abstract

Procédés de détection et/ou de dénombrement des contaminants se trouvant dans des échantillons tels que des échantillons pharmaceutiques et des échantillons de denrées alimentaires, de boissons, et d'eau. Les procédés associent la spécificité de l'immunodétection à la vitesse des systèmes automatisés afin d'obtenir des procédés rapides de détection des contaminants spécifiques dans les échantillons.
PCT/AU1991/000247 1990-06-08 1991-06-07 Procede de detection WO1991019003A1 (fr)

Applications Claiming Priority (2)

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AUPK056690 1990-06-08
AUPK0566 1990-06-08

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WO1991019003A1 true WO1991019003A1 (fr) 1991-12-12

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NZ (1) NZ238444A (fr)
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027144A1 (fr) * 1993-05-19 1994-11-24 California South Pacific Investors Detection de contaminants dans les aliments
GB2285863A (en) * 1993-12-02 1995-07-26 Inia Immunoassay for cellular organisms
US5635617A (en) * 1993-04-26 1997-06-03 University Of Victoria Innovation & Development Corp. Methods and compositions comprising the agfA gene for detection of Salmonella
AT409190B (de) * 1999-01-18 2002-06-25 Helmut Dr Pfuetzner Kontaminationswächter
EP1440316A4 (fr) * 2001-10-10 2006-08-16 Tecra Internat Pty Ltd Systeme et appareil de detection de micro-organismes
CN111471592A (zh) * 2020-05-11 2020-07-31 华中科技大学同济医学院附属协和医院 一种细胞培养瓶及其制作方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4637185A (en) * 1984-08-01 1986-02-25 Advanced Biotechnology Associates Inc. A salmonella specific analysis reagent
EP0214340A2 (fr) * 1985-09-09 1987-03-18 BioControl Systems, Inc. Procédé de détection d'organismes mobiles, sélectionnés
AU2127888A (en) * 1987-07-28 1989-03-01 Tecra International Pty Ltd Detection methods

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE430900B (sv) * 1981-06-04 1983-12-19 Bo Gustav Mattiasson Forfarande for bestemning av biokemiska data for mikroorganismer eller encelliga organismer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4637185A (en) * 1984-08-01 1986-02-25 Advanced Biotechnology Associates Inc. A salmonella specific analysis reagent
EP0214340A2 (fr) * 1985-09-09 1987-03-18 BioControl Systems, Inc. Procédé de détection d'organismes mobiles, sélectionnés
AU2127888A (en) * 1987-07-28 1989-03-01 Tecra International Pty Ltd Detection methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0533772A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635617A (en) * 1993-04-26 1997-06-03 University Of Victoria Innovation & Development Corp. Methods and compositions comprising the agfA gene for detection of Salmonella
WO1994027144A1 (fr) * 1993-05-19 1994-11-24 California South Pacific Investors Detection de contaminants dans les aliments
GB2285863A (en) * 1993-12-02 1995-07-26 Inia Immunoassay for cellular organisms
GB2285863B (en) * 1993-12-02 1998-01-28 Inia Method for the detection of Erwinia carotovora atroseptica subspecies by "ELISA-Enrichment"
AT409190B (de) * 1999-01-18 2002-06-25 Helmut Dr Pfuetzner Kontaminationswächter
EP1440316A4 (fr) * 2001-10-10 2006-08-16 Tecra Internat Pty Ltd Systeme et appareil de detection de micro-organismes
US7374951B2 (en) 2001-10-10 2008-05-20 Tecrainternational Pty Ltd. System and apparatus for use in detecting microorganisms
EP2128620A1 (fr) * 2001-10-10 2009-12-02 3M Innovative Properties Company Système et appareil pour une utilisation dans la détection de micro-organismes
US7771986B2 (en) 2001-10-10 2010-08-10 3M Innovative Properties Company System and apparatus for use in detecting microorganisms
CN111471592A (zh) * 2020-05-11 2020-07-31 华中科技大学同济医学院附属协和医院 一种细胞培养瓶及其制作方法

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EP0533772A1 (fr) 1993-03-31
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